Chimeric receptor genes and cells transformed therewith

ABSTRACT

Chimeric receptor genes suitable for endowing lymphocytes with antibody-type specificity include a first gene segment encoding a single-chain Fv domain of a specific antibody and a second gene segment encoding all or part of the transmembrane and cytoplasmic domains, and optionally the extracellular domain, of an immune cell-triggering molecule. The chimeric receptor gene, when transfected to immune cells, expresses the antibody-recognition site and the immune cell-triggering moiety into one continuous chain. The transformed lymphocytes are useful in therapeutic treatment methods.

[0001] This application is a continuation in part of U.S. applicationSer. No. 08/084,994 filed Jul. 2, 1993, which is herein incorporated byreference in toto.

FIELD OF THE INVENTION

[0002] The present invention relates to chimeric receptor genes suitablefor endowing lymphocytes with antibody-type specificity, to expressionvectors comprising said chimeric genes and to lymphocytes transformedwith said expression vectors. Various types of lymphocyte cells aresuitable, for example, cytotoxic T cells, helper T cells, natural killer(NK) cells, etc. The transformed lymphocytes are useful in therapeutictreatment methods.

BACKGROUND OF THE INVENTION

[0003] Cells of the immune system are known to recognize and interactwith specific molecules by means of receptors or receptor complexeswhich, upon recognition or an interaction with such molecules, causesactivation of the cell to perform various functions. An example of sucha receptor is the antigen-specific T cell receptor complex (TCR/CD3).

[0004] The T cell receptor for antigen (TCR) is responsible for therecognition of antigen associated with the major histocompatibilitycomplex (MHC). The TCR expressed on the surface of T cells is associatedwith an invariant structure, CD3. CD3 is assumed to be responsible forintracellular signalling following occupancy of the TCR by ligand.

[0005] The T cell receptor for antigen-CD3 complex (TCR/CD3) recognizesantigenic peptides that are presented to it by the proteins of the majorhistocompatibility complex (MHC). Complexes of MHC and peptide areexpressed on the surface of antigen presenting cells and other T celltargets. Stimulation of the TCR/CD3 complex results in activation of theT cell and a consequent antigen-specific immune response. The TCR/CD3complex plays a central role in the effector function and regulation ofthe immune system.

[0006] Two forms of T cell receptor for antigen are expressed on thesurface of T cells. These contain either α/β heterodimers or γ/δheterodimers. T cells are capable of rearranging the genes that encodethe α, β, γ and δ chains of the T cell receptor. T cell receptor generearrangements are analogous to those that produce functionalimmunoglobulins in B cells and the presence of multiple variable andjoining regions in the genome allows the generation of T cell receptorswith a diverse range of binding specificities. Each α/β or γ/δheterodimer is expressed on the surface of the T cell in associationwith four invariant peptides. These are the γ, Ε and ε subunits of theCD3 complex and the zeta chain. The CD3 γ, δ and ε polypeptides areencoded by three members of the immunoglobulin supergene family and arefound in a cluster on human chromosome 11 or murine chromosome 9. Thezeta chain gene is found separately from other TCR and CD3 genes onchromosome 1 in both the mouse and human. Murine T cells are able togenerate a receptor-associated η chain through alternative splicing ofthe zeta m-RNA transcript. The CD3 chains and the zeta subunit do notshow variability, and are not involved directly in antigen recognition.

[0007] All the components of the T cell receptor are membrane proteinsand consist of a leader sequence, externally-disposed N-terminalextracellular domains, a single membrane-spanning domain, andcytoplasmic tails. The α, β, γ and δ antigen-binding polypeptides areglycoproteins. The zeta chain has a relatively short ectodomain of onlynine amino acids and a long cytoplasmic tail of approximately 110 aminoacids. Most T cell receptor α/β heterodimers are covalently linkedthrough disulphide bonds, but many γ δ receptors associate with oneanother non-covalently. The zeta chain quantitatively forms eitherdisulphide-linked ζ-η heterodimers or zeta-zeta homodimers.

[0008] Another example of a type of receptor on cells of the immunesystem is the Fc receptor. The interaction of antibody-antigen complexeswith cells of the immune system results in a wide array of responses,ranging from effector functions such as antibody-dependent cytotoxicity,mast cell degranulation, and phagocytosis to immunomodulatory signalssuch as regulating lymphocyte proliferation, phagocytosis and targetcell lysis. All these interactions are initiated through the binding ofthe Fc domain of antibodies or immune complexes to specialized cellsurface receptors on hematopoietic cells. It is now well establishedthat the diversity of cellular responses triggered by antibodies andimmune complexes results from the structural heterogeneity of Fcreceptors (FcRs).

[0009] FcRs are defined by their specificity for immunoglobulinisotypes. Fc receptors for IgG are referred to as FcγR, for IgE as FcεR,for IgA as FcαR, etc. Structurally distinct receptors are distinguishedby a Roman numeral, based on historical precedent. We now recognizethree groups of FcγRs, designated FcγRI, FcγRII, and FcγRIII. Two groupsof FcεR have been defined; these are referred to as FcεRI and FcεRII.Structurally related although distinct genes within a group are denotedby A, B, C. Finally, the protein subunit is given a Greek letter, suchas FcγRIIIAα, FcγRIIIAγ.

[0010] Considerable progress has been made in the last three years indefining the heterogeneity for IgG and IgE Fc receptors (FcγR, FcεR)through their molecular cloning. Those studies make it apparent that Fcreceptors share structurally related ligand binding domains, but differin their transmembrane and intracellular domains which presumablymediate intracellular signalling. Thus, specific FcγRs on differentcells mediate different cellular responses upon interaction with animmune complex. The structural analysis of the FcγRs and FcεRI has alsorevealed at least one common subunit among some of these receptors. Thiscommon subunit is the γ subunit, which is similar to the ζ or η chain ofthe TCR/CD3, and is involved in the signal transduction of the FcγRIIIand FcεRI.

[0011] The low affinity receptor for IgG (FcγRIIIA), is composed of theligand binding CD16α (FcγRIIIAα) polypeptide associated with the γ chain(FcγRIIIAγ). The CD16 polypeptide appears as membrane anchored form inpolymorphonuclear cells and as transmembrane form (CD16_(TM)) in NK. TheFcγRIIIA serves as a triggering molecule for NK cells.

[0012] Another type of immune cell receptor is the IL-2 receptor. Thisreceptor is composed of three chains, the α chain (p55), the β chain(p75) and the γ chain. When stimulated by IL-2, lymphocytes undergoproliferation and activation.

[0013] Antigen-specific effector lymphocytes, such as tumor specific Tcells (Tc), are very rare, individual-specific, limited in theirrecognition spectrum and difficult to obtain against most malignancies.Antibodies, on the other hand, are readily obtainable, more easilyderived, have wider spectrum and are not individual-specific. The majorproblem of applying specific antibodies for cancer immunotherapy lies inthe inability of sufficient amounts of monoclonal antibodies (mAb) toreach large areas within solid tumors. In practice, many clinicalattempts to recruit the humoral or cellular arms of the immune systemfor passive anti-tumor immunotherapy have not fulfilled expectations.While it has been possible to obtain anti-tumor antibodies, theirtherapeutic use has been limited so far to blood-borne tumors (1, 2)primarily because solid tumors are inaccessible to sufficient amounts ofantibodies (3). The use of effector lymphocytes in adoptiveimmunotherapy, although effective in selected solid tumors, suffers onthe other hand, from a lack of specificity (such as in the case oflymphokine-activated killer cells (LAK cells) (4) which are mainly NKcells) or from the difficulty in recruiting tumor-infiltratinglymphocytes (TILs) and expanding such specific T cells for mostmalignancies (5). Yet, the observations that TILs can be obtained inmelanoma and renal cell carcinoma tumors, that they can be effective inselected patients and that foreign genes can function in these cells (6)demonstrate the therapeutic potential embodied in these cells.

[0014] A strategy which has been recently developed (European PublishedPatent Application No. 0340793, Ref. 7-11) allows one to combine theadvantage of the antibody's specificity with the homing, tissuepenetration, cytokine production and target-cell destruction of Tlymphocytes and to extend, by ex vivo genetic manipulations, thespectrum of anti-tumor specificity of T cells. In this approach thelaboratory of the present inventors succeeded to functionally express inT cells chimeric T cell receptor (cTCR) genes composed of the variableregion domain (Fv) of an antibody molecule and the constant regiondomain of the antigen-binding TCR chains, i.e., the α/β or γ/δ chains.In this gene-pairs approach, genomic expression vectors have beenconstructed containing the rearranged gene segments coding for the Vregion domains of the heavy (V_(H)) and light (V_(L)) chains of ananti-2,4,6-trinitrophenyl (TNP) antibody (Sp6) spliced to either one ofthe C-region gene segments of the α or β TCR chains. Followingtransfection into a cytotoxic T-cell hybridoma, expression of afunctional TCR was detected. The chimeric TCR exhibited the idiotope ofthe Sp6 anti-TNP antibody and endowed the T cells with a majorhistocompatibility complex (MHC) nonrestricted response to the haptenTNP. The transfectants specifically killed TNP-bearing target cells, andproduced interleukin-2 (IL-2) in response thereto across strain andspecies barriers. Moreover, such transfectants responded to immobilizedTNP-protein conjugates, bypassing the need for cellular processing andpresentation. The chimeric TCRs could provide T cells with anantibody-like specificity and, upon encountering antigen, were able toeffectively transmit signals for T cell activation, secretion oflymphokines and specific target cell lysis in a MHC nonrestrictedmanner. Moreover, the cTCR bearing cells undergo stimulation byimmobilized antigen, proving that receptor-mediated T-cell activation isnot only nonrestricted but also independent of MHC expression on targetcells (8, 9). New expression cassettes were also developed based onreverse transcription of mRNA and PCR amplification of rearranged V_(H)and V_(L) DNA, using primers based on 3′ and 5′ consensus sequences (12)of these genes which allow rapid construction of cTCR genes from anymAb-producing hybridoma. To determine the therapeutic potential of thechimeric TCR approach, we successfully constructed and functionallyexpressed cTCR genes composed of combining sites of anti-idiotypicantibody specific to the surface IgM of the 38C13 murine B lymphoma cellline.

[0015] Broad application of the cTCR approach is dependent on efficientexpression of the cTCR genes in primary T cells. So far, utilizingprotoplast fusion, lipofection or electroporation, we succeeded inexpressing the cTCR in T cell hybridomas (8, 9) or human T cell tumors,such as Jurkat, but like others, achieved only limited and transientexpression in non-transformed murine T cell lines. Although retroviralvectors have been demonstrated to be effective for transgene expressionin human T cells (13, 14), due to the fact that two genes have to beintroduced in order to express functional cTCR; (CαV_(H)+CβV_(L) orCαV_(L)+CβV_(H)), and the very low efficiency of transduction of asingle cell with two separate retroviral vectors, new vectors have to betried which will allow the transduction of two genes in tandem (15).

[0016] Another strategy which has recently been developed employsjoining of the extracellular ligand binding domain of receptors such asCD4, CD8, the IL-2 receptor, or CD16, to the cytoplasmic tail of eitherone of the γ/ζ family members (26-28, 38). It has been shown thatcrosslinking of such extracellular domains through a ligand or antibodyresults in T cell activation. Chimeric CD4 or CD16-γ/ζ moleculesexpressed in cytotoxic lymphocytes could direct specific cytolysisagainst appropriate target cells (26, 38). In PCT WO92/15322 it issuggested that the formation of chimeras consisting of the intracellularportion of T cell/Fc receptor ζ, ε or γ chains joined to theextracellular portion of a suitably engineered antibody molecule willallow the target recognition potential of an immune system cell to bespecifically redirected to the antigen recognized by the extracellularantibody portion. However, while specific examples are present showingthat such activation is possible when the extracellular portion ofreceptors such as the CD4 receptor are joined to such ζ, η or γ chains,no proof was presented that when a portion of an antibody is joined tosuch chains one can obtain expression in lymphocytes or activation oflymphocytes.

SUMMARY OF THE INVENTION

[0017] It has now been found according to the present invention that byfusing a single-chain Fv domain (scFv) gene of a specific antibody,composed of V_(L) linked to V_(H) by a flexible linker, with a genesegment encoding a short extracellular and the entire transmembrane andcytoplasmic domains of a lymphocyte-activation molecule, a chimeric geneis obtained which combines the antibody recognition site and thelymphocyte-signalling moiety into one continuous chain. Upontransfection of such chimeric scFv-receptor (c-scFvR) gene intolymphocytes, it is expressed in the cell as a functional receptor andendows the cells with antibody-type specificity.

[0018] The present invention thus relates to chimeric genes suitable toendow lymphocyte cells with antibody-type specificity. Various types oflymphocytes are suitable, for example, natural killer cells, helper Tcells, suppressor T cells, cytotoxic T cells, lymphokine activatedcells, subtypes thereof and any other cell type which can expresschimeric receptor chain.

[0019] The chimeric gene comprises a first gene segment encoding thescFv of a specific antibody, i.e., DNA sequences encoding the variableregions of the heavy and light chains (V_(H) and V_(L), respectively) ofthe specific antibody, linked by a flexible linker, and a second genesegment which comprises a DNA sequence encoding partially or entirelythe transmembrane and cytoplasmic, and optionally the extracellular,domains of a lymphocyte-triggering molecule corresponding to alymphocyte receptor or part thereof.

[0020] The present invention further relates to suitable vectors fortransfecting cells of the type defined above with the chimeric gene.

[0021] The present invention further relates to cells of the typedefined above into which such chimeric gene has been introduced so as toobtain its expression, and also to pharmaceutical prophylactic andcurative compositions containing an effective quantity of such cells.

[0022] In general terms, the present invention relates to a process forthe generation of lymphocytes transfected with an expression vectorcontaining a chimeric gene of the invention. As set out in thefollowing, there was constructed a model system which comprises anexpression vector which was transfected into cytotoxic T cells and whichwas functionally expressed in said cells, i.e., which directed thecellular response of the lymphocyte against a predefined target antigenin a MHC nonrestricted manner.

[0023] The genetically engineered lymphocyte cells of the presentinvention may be used in new therapeutic treatment processes. Forexample, T cells or NK cells isolated from a patient may be transfectedwith DNA encoding a chimeric gene including the variable region of anantibody directed toward a specific antigen, and then returned to thepatient so that the cellular response generated by such cells will betriggered by and directed toward the specific antigen in a MHCnonrestricted manner. In another embodiment, peripheral blood cells ofthe patient are genetically engineered according to the invention andthen administered to the patient.

[0024] Because of the restrictions imposed by corecognition of self MHCplus antigen, the acquisition of new specificity by grafting of TCRgenes is limited to inbred combinations. Such manipulations arepractically impossible in an outbred population. However, the presentinvention allows us to confer antibody specificity using not only theTCR components, but other lymphocyte-signalling chains, such as thezeta/eta chains of CD3, γ chain of the FcγR and FcεR, α, β and γ ψchains of the IL-2R or any other lymphokine receptor, CD16 α-chain, CD2,CD28, and others. Thus, grafting the chimeric genes into NK cells whichare not antigen-specific will endow them with antibody specificity.

DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1 depicts a scheme of the chimeric scFvR expression vector. Rrepresents any receptor chain, such as the zeta subunit of the CD3,gamma and CD16α subunits of the FCγRIII, Cα and Cβ of the TCR, β chainof the IL-2 receptor or any other chain or part thereof describedherein. A depicts the preparation of the gene segments encoding the scFvof the V_(H) and V_(L) of a specific antibody linked by a flexiblelinker (hatched box). B represents the pRSV expression vector containingthe kappa light chain leader (L_(κ)), into which the receptor geneprepared from lymphocytes described in C and the gene segment of A areintroduced. Expression of the chimeric gene is driven by the longterminal repeat (LTR) promoter of the Rous sarcoma virus.

[0026]FIG. 2 illustrates the chimeric pRSVscFvRγ expression vectorobtained according to the scheme of FIG. 1. The boxes from left to rightrepresent DNA segments corresponding to the Rous sarcoma virus longterminal repeat promoter (LTR), kappa light chain leader (L_(κ)) andvariable region (V_(κ)), the linker (hatched box), heavy chain variableregion (V_(H)) the human gamma chain, the G418-resistance gene (neo^(r))and the simian virus 40 origin of replication. Restriction sitesindicated are EcoRI (RI), SnaBI (Sn), NcoI (N), XbaI (Xb), SalI (S),BstEII (Bs), and XhoI (X). The arrowheads numbered 1 to 6 represent theflanking regions amplified by using the oligonucleotide primers 4, 5, 6,7, 14 and 15, respectively shown in Table I, infra.

[0027] These primers were designed to match the consensus sequences ofV_(H) and V_(L). The relevant restriction sites are in bold letters.

[0028]FIG. 3 shows the fluorescence-activated cell sorter (FACS)analysis of immunofluorescence staining of MD.45 hybridoma and its TCRα-MD45.27J mutant, their corresponding scFvRγ-transfected STA and STBclones, or STZ cells, which result from transfection of the scFvRζchimeric gene into MD45.27J. Solid line, staining with anti-Sp6idiotypic antibody 20.5 or anti-CD3 mAb 145.2Cll. Broken line representscontrol irrelevant antibody.

[0029] FIGS. 4A-4D show immunoblotting analysis of lysates prepared fromscFvRγ transfectants and parental hybridomas developed by anti-Sp6idiotypic mAb 20.5 (FIGS. 4A and 4C, respectively) and rabbit anti-humangamma chain (FIGS. 4B and 4D, respectively). Electrophoresis was on fourseparate gels. The molecular mass scales are related to B and D; thearrows point to the same bands in A and B or C and D.

[0030]FIGS. 5A and 5B show the composition of the scFvRγ dimers. FIG.5A—Immunoblot analysis of anti-Sp6 precipitates prepared from STB(scFvRγ transfectant cells), and their parent (MD45.27J hybridomacells). After electrophoresis under non-reducing conditions andblotting, the blot was allowed to react with anti-Sp6, anti-human gamma,or anti-mouse ζ antibodies. FIG. 5B—Immunoprecipitation of lysates madeof surface-iodinated STB cells (scFvRγ transfectant cells) and theirparent (MD45.27J hybridoma cells).

[0031] FIGS. 6A-6B illustrates that transfectants expressing scFvR arestimulated to produce IL-2 after stimulation with TNP-A.20 (FIG. 6A), orplastic immobilized TNP-FγG, without or with different concentrations ofsoluble TNP-FγG (FIG. 6B). GTAc.20 is an SpG double-chain cTCRtransfectant described previously (9). The scFvR zeta-expressing STZproduced about 200 units (U) of IL-2 per ml after co-culture withTNP-A.20 at 8:1 stimulator-to-effector (S/E) cell ratio. Not shown arethe responses of the transfectants to non-modified A.20 or FγG controls,which were completely negative, exactly like the background responses ofthe MD.45 and MD45.27J to TNP antigen.

[0032]FIGS. 7A and 7B show specific ⁵¹Cr release of TNP-A.20 cells afterincubation with transfectants expressing scFvR. Effector cells wereincubated with plastic-immobilized TNP-FγG for 8 hr before the killingassay. Kinetic assay was done at an effector-to-target (E/T) cell ratioof 10:1 (FIG. 7A); dose response was determined in a 9 hr assay (FIG.7B). Control non-modified A.20 target cells incubated with the sameeffector cells in identical conditions did not release more ⁵¹Cr thanthe spontaneous release (not shown).

[0033] FIGS. 8A-8D show surface expression of chimeric scFvRγ/ζ. T cellhybridoma transfected with the scFvRγ (N29γ1, N29γ15) or scFvRζ(N29ζM.1) chimeric genes composed of the variable region of N29anti-HER2 mAb, were stained with anti-N29 idiotypic antibodies orcontrol serum (broken lines) and analyzed by FACS.

[0034]FIGS. 9A and 9B show binding of detergent-solubilized scFvN29Rγand scFvN29Rζ to Neu/HER2 antigen. The presence of chimeric receptors incell lysates was evaluated by ELISA using HER2X-coated wells and anti-γ(FIG. 9A) or anti-ζ (FIG. 9B) antibodies. Functional molecules derivedfrom hybridomas expressing the chimeric transgenes could bind to theimmobilized antigen and expressed antigenic determinants specific toeither γ or ζ polypeptides.

[0035]FIGS. 10A and 10B shows antigen-specific activation of chimericreceptor expressing cells by HER2-bearing stimulator cells (FIG. 10A) orimmobilized HER2X protein (FIG. 10B). T cell hybridomas expressing thechimeric scFvN29Rγ/ζ genes underwent antigen-specific, but MHCunrestricted stimulation for IL-2 production following co-culture witheither HER2-expressing cells of different origins or with plastic-boundpurified HER2/Neu receptor. Stimulator cells used were human breastcarcinoma cell lines SKBR3 and MDA 468, the human ovarian carcinoma cellline SKOV3 or HER2, a c-erbB-2 transfected 3T3-NIH fibroblasts (kindlyprovided by Dr. A. Ullrich). The Neu/HER2 protein is overexpressed inSKBR3, SKOV3 and HER2, while the MDA 468 cells have undetectable surfacereceptor. As shown, untransfected parental cells MD45.27J did notproduce any IL-2 following incubation with Neu/HER2 expressing cells. InB, [filled square]—MD45.27J, untransfected cells; O—N29γ1, transfectantexpressing scFvN29Rγ.

[0036]FIG. 11 shows that chimeric receptor expressing cells specificallylyse Neu/HER2 target cells. Non-transfected CTL hybridomas and thescFvN29Rγ expressing (N29γ1) or the scFvN29Rζ expressing (MD45ζ1)transfectants were studied for their cytolytic potential either towardNeu/HER2 expressing NIH-3T3 murine fibroblasts or the human colon (N87)or breast (SKBR3) carcinoma cell lines. The percent ⁵¹Cr released by theparental cells at the same E:T were subtracted.

[0037]FIG. 12 shows that chimeric receptor expressing cells specificallylyse HER2 target cells. Non-transfected CΓL hybridomas and the scFvN29Rγexpressing (N29γ1) or the scFvN29Rζ expressing (N29ζ18) transfectantswere studied for their cytolytic potential either toward Neu/HER2expressing NIH-3T3 murine fibroblasts (filled symbols) or thenon-transfected NIH-3T3 cells (open symbols). Substantial and specificlysis of HER2 target cells was demonstrated by N29γ1 at all effector totarget (E:T) ratios. Weak lysis of HER2 as compared to the untransfectedfibroblasts was observed for N29ζ18, while the MD45 and MD45.27J,non-transfected hybridomas did not cause any significant ⁵¹Cr release.[filled triangle], [empty triangle], -N29γ1; [filled circle]-N29ζ18;[filled square], [empty square]-MD45.27J.

[0038]FIG. 13 shows transfer of the scFvR gene from the pRSVneo-scFvR tothe pBJ1-neo vector. The scFvR was cut out from the pRSV vector usingthe SnaBI and introduced into the EcoRV site of the polylinker of thepBJ1 plasmid to drive the expression of the chimeric gene from the SRαpromoter.

[0039] FIGS. 14A-14E. FIG. 14A shows schematic representation of rosetteformation by T cells expressing the anti-IgE scFvCβ chimeric gene. Sheepred blood cells (HRBC) were coated with TNP and then with anti-TNP ofthe IgE class. The IgE-TNP-SRBC- complex was incubated with the T cellstransfected with the scFvR comprising the scFv of the anti-IgE 84-1cmAb, and observed under microscope for rosette formation. FIG. 14B showsresults of the rosette formation on scFvR-transfected JRT.T3.5 cells.parental JRT.T3.5 cells were used as negative and the 84.1c as positivecontrols. Results are given in percentage of cells that form rosettes.FIG. 14C shows inhibition, of rosette formation of transfectantsexpressing scFvR. The transfectants were incubated with IgE, anti-Fc andanti-MYC, IgG (as negative control) and then with the SRBC-conjugate andcounted. FIG. 14D shows rosette formation of the JSB.15 transfectant.FIG. 14E shows rosette formation of the MD.45 derived transfectantsexpressing the scFvR.MD.45 was used as negative control.

[0040] FIGS. 15A-15B. FIG. 15A shows schematic representation of theELISA used to screen transfectants expressing scFvCβ chimeric gene (R isCβ). Plates were covered with IgE and lysates of the transfectants wereadded, then anti-human α/β TCR antibodies were added and the reactionwas developed with goat anti-mouse peroxidase. FIG. 15B shows results ofsome transfectants expressing the scFvR in the ELISA anti-human β TCRantibodies.

[0041]FIG. 16 shows stimulation of transfectants with immobilized IgE oranti-CD3 for IL-2 production. plates were coated with 2.5 μg/ml ofeither IgE or anti-CD3 purified antibodies and transfectant cells wereincubated in the presence of phorbol 12-mirystate 13-acetate (PMA), (10ng/ml) for 20-24 hours. supernatants were collected and IL-2 productionwas determined using the IL-2 dependent cell line CTLL. UntransfectedJRT.T3.5 cell was used as negative control and controls for thedifferent media were also included in the CTLL assay.

[0042]FIG. 17 shows stimulation for IL-2 production with IgE positive Bcells. The SPE-7 IgE secretor hybridoma was fixed with 0.25%glutaraldehyde for 10 min. at 0° C. and mixed with the transfectants indifferent effector/stimulator (E/S) ratio. Cells were incubated for20-24 hours and supernatants were collected and assayed for IL-2production.

[0043]FIGS. 18A and 18B show specific inhibition of IgE production bycytotoxic hybridoma expressing the anti-IgE scFvR. Spleen cells werestimulated with 20 μg/ml LPS and 100 U/ml IL-4 for four days. At day 4spleen cells were washed and MD.45 cytotoxic hybridoma expressing thescFv was added and IgE and IgG concentrations were measured after 24, 48and 72 hours. 84.1c hybridoma cells were included as control as well asthe MD.45.

[0044]FIG. 19 is a schematic representation of the chimeric scFv-CD16gene.

[0045] FIGS. 20A-20D show surface staining of rat basophilic leukemia(RBL) cells transfected with the scFvCD16 gene. Immunofluorescencestaining was performed with anti-Sp6 idiotypic mAb 20.5 and irrelevantmouse antibody as negative control. The shift to the right in the FACSstaining pattern is due to chimeric receptor expressing cells.

[0046] FIGS. 21A-21D show surface staining of RBL cells transfected withscFvRγ or scFvRζ chimeric genes. Immunofluorescence staining wasperformed with anti-Sp6 idiotypic mAb 20.5 and irrelevant mouse antibodyas negative control.

[0047]FIGS. 22A and 22B show surface staining of murine thymoma BW5147cells transfected with the scFvCD16 gene. Immunofluorescence stainingwas performed with anti-Sp6 idiotypic mAb 20.5 and irrelevant mouseantibody as negative control.

[0048]FIG. 23 shows stimulation of BW5147 cells co-transfected withscFvCD16 and normal γ chain by TNP-labeled A.20 target cells.BW-scFvCD16 clone 45 (A) or clone 50 (B) were co-cultured at differenttarget: stimulation ratios with TNP modified irradiated A.20 cells. IL-2produced into the supernatant was determined following 24 hours by theMTT assay.

[0049]FIGS. 24A and 24B show stimulation of BW5147 cells cotransfectedwith scFvCD16 and normal γ chain by immobilized TNP-Fowl γ-globulin(TNP-FγG). Different concentrations of TNP-FγG at different TNP:FγGratios were used to coat the wells of a microculture plate. IL-2 wasdetermined in the supernatant of 24 hr cultures of BW-scFvCD16 clone 5(FIG. 24A) or clone 50 (FIG. 24B). Incubation of either one of the cellswith immobilized FγG by itself (filled squares) did not stimulate thecells. The parental BW cells did not make any IL-2 in response toTNP-FγG under the same conditions (not shown).

[0050] FIGS. 25A-25D show surface staining of RBL cells transfected withthe scFvIL2R gene. Immunofluorescence staining was performed withanti-Sp6 idiotypic mAb 20.5 and irrelevant mouse antibody as negativecontrol.

[0051] FIGS. 26A-26D show that BW5147 cells transfected with scFvRexpress surface chimeric receptors. BW5147 cells transfected withSp6-scFvR were reacted with 1:200 dilution of ascites of 20.5 anti-Sp6idiotypic antibody or anti-MOv18 ascites in the same dilution ascontrol, followed by FITC labeled anti-mouse Ig. Immunofluorescence wasdetected by FACS. BW.Sp6-CD16 are cells co-transfected with scFvCD16 andγ chain. Cells transfected with scFvCD16 alone did not stain above theuntransfected BW cells.

[0052]FIG. 27 shows stimulation of scFvR-BW5147 transfectants withTNP-A.20 cells. Different BW-scFvR transfectants were incubated withvarious amounts of TNP-A.20 cells for 24 hrs. IL-2 was determined by theMTT calorimetric assay. BWG are scFvRγ transfectants and BWZ are scFvRζtransfectants.

[0053]FIG. 28 shows that scFvR transfected BW5147 cells respond toimmobilized TNP. Different BW-scFvR transfectants ware incubated withTNP₁₅-FγG coated wells for 24 hrs. IL-2 was determined by the MTTcalorimetric assay. The abscissa describes the concentrations of TNP-FγGused to coat the wells of a microtitre plate. BWG are scFvRζtransfectants and BWZ are scFvRζ transfectants.

DETAILED DESCRIPTION OF THE INVENTION

[0054] To overcome the difficulties of the prior method involving thegene-pairs approach (the “T-body” approach) and to extend itsapplicability to other cells and receptor molecules, a new alternativedesign was developed according to the invention. It is based on asingle-chain approach to the cTCR and on the demonstrated ability toexpress in bacteria an antibody single-chain Fv domain (scFv) (16, 17).Such scFv domains, which join the antibody's heavy and light variable(V_(H) and V_(L)) gene segments with a flexible linker, have proven toexhibit the same specificity and affinity as the natural Fab′ fragment.Thus, one immediate application of the scFv is to construct chimericmolecules composed of scFv linked to one of the TCR constant domains.

[0055] According to the invention, chimeric molecules were constructedcomposed of the scFv linked to receptor subunits that might serve totransduce the signal from the scFv and confer antibody specificity to Tcells as well as other lymphocytes. This construction is preferablyaccomplished in the manner shown in FIG. 1 at A, DNA or RNA fromantibody forming cells is isolated. cDNA is prepared from mRNA andamplification of the antibody light and heavy variable regions (V_(H)and V_(L)) by PCR using a V_(L) -5′ (XbaI) , V_(L)-3 (SalI), V_(H)-5′(SalI) and V_(H)-3′ (BstEII) specific primers. As shown at B, To thepRSV₂-neo plasmid a leader sequence from the S1C5 kappa chain wasintroduced down stream from the LTR promoter. At C, RNA from Tlymphocytes was isolated and from the cDNA prepared the α, β chains ofthe TCR, γ, ζ subunits of the CD3, CD16α of the FCγRIII, or IL-2receptors (commonly denoted here as R) can be amplified using a specificset of primers for each chain. All the primers include a XbaI at their5′ end and a few bases downstream of the XbaI or the BstEII site. At the3′ end, all receptor chains contain a SnaBI site. Following introductionof the leader sequence into the pRSV₂-neo plasmid the receptor wasintroduced at the XbaI site of the pRSVneoL_(κ) vector obtainingpRSVneoL_(κ)-R. The amplified V_(L) (digested with XBaI-SalI) and V_(H)(digested with SalI-BstEII) regions are introduced into the XbaI-BstEIIdigested pRSVneoL_(κ)-R plasmid in a three-piece ligation. The resultingplasmid pRSVscFvR contains the complete chimeric single chain receptor.The receptor (R) gene segment described in FIGS. 13-18 is the human TCRCβ.

[0056] Thus, the new strategy according to the invention enables the useof other receptor molecules which might serve to transduce the signalfrom the scFv and confer antibody specificity to T cells as well asother immune cells. In fact, it allows the expression of the scFv as theantigen recognition unit of chimeric molecules composed of thetransmembrane and cytoplasmic domains of receptor molecules of immunecells, such as T cells and natural killer (NK) cells. Such receptors canbe single or multi-chain in nature and not necessarily belong to the Iggene superfamily.

[0057] Candidate molecules for this approach are receptor moleculeswhich take part in signal transduction as an essential component of areceptor complex, such as receptors which trigger T cells and NKactivation and/or proliferation. Examples of triggers of T cells aresubunits of the TCR, such as the α, β, γ or δ chain of the TCR, or anyof the polypeptides constituting the CD3 complex which are involved inthe signal transduction, e.g., the γ, δ, ε, ζ and η CD3 chains. Amongthe polypeptides of the TCR/CD3 (the principal triggering receptorcomplex of T cells), especially promising are the zeta and its etaisoform chain, which appear as either homo- or hetero-S—S-linked dimers,and are responsible for mediating at least a fraction of the cellularactivation programs triggered by the TCR recognition of ligand (18, 19).These polypeptides have very short extracellular domains which can servefor the attachment of the scFv.

[0058] Additional examples of immune cell trigger molecules are any oneof the IL-2 receptor (IL-2R) p55 (α) or p75 (β) or γ chains, especiallythe p75 and γ subunits which are responsible for signaling T cell and NKproliferation.

[0059] Further candidate receptor molecules for creation of scFvchimeras in accordance with the present invention include the subunitchains of Fc receptors.

[0060] In the group of NK-stimulatory receptors the most attractivecandidates are the γ- and CD16α-subunits of the low affinity receptorfor IgG, FcγRIII. Occupancy or cross-linking of FcγRIII (either byanti-CD16 or through immune complexes) activates NK cells for cytokineproduction, expression of surface molecules and cytolytic activity (20,21). In NK cells, macrophages, and B and T cells, the FcγRIII appears asa heterooligomeric complex consisting of a ligand-binding α chainassociated with a disulfide-linked γ or zeta chain. The FcγRIIIAsignalling gamma chain (22) serves also as part of the FcεRI complex,where it appears as a homodimer, is very similar to the CD3 zeta chain,and in fact can form heterodimers with it in some cytolytic Tlymphocytes (CTL) and NK cells (23-25). Most recently prepared chimerasbetween these polypeptides and the CD4 (26), the CD8 (27), IL-2 receptorchain (28) or CD16 extracellular domains, proved to be active insignalling T cell stimulation even in the absence of other TCR/CD3components.

[0061] In addition to the receptor molecules discussed above, there arelymphocyte accessory and adhesion molecules such as CD2 and CD28, whichtransduce a co-stimulatory signal for T-cell activation. Theseco-stimulatory receptors can also be used in accordance with the presentinvention.

[0062] Besides the specific receptor chains specifically mentionedherein, the single chain Fv chimeras can be made by joining the scFvdomain with any receptor or co-receptor chain having a similar functionto the disclosed molecules, e.g., derived from granulocytes, Blymphocytes, mast cells, macrophages, etc. The distinguishing featuresof desirable immune cell trigger molecules comprise the ability to beexpressed autonomously (i.e., as a single chain), the ability to befused to an extracellular domain such that the resultant chimera isexpressed on the surface of an immune cell into which the correspondinggene was genetically introduced, and the ability to take part in signaltransduction programs secondary to encounter with a target ligand.

[0063] The scFv domain must be joined to the immune cell triggeringmolecule such that the scFv portion will be extracellular when thechimera is expressed. This is accomplished by joining the scFv either tothe very end of the transmembrane portion opposite the cytoplasmicdomain of the trigger molecule or by using a spacer which is either partof the endogenous extracellular portion of the triggering molecule orfrom other sources. The chimeric molecules of the present invention havethe ability to confer on the immune cells on which they are expressedMHC nonrestricted antibody-type specificity. Thus, a continuouspolypeptide of antigen binding and signal transducing properties can beproduced and utilized as a targeting receptor on immune cells. In vivo,cells expressing these genetically engineered chimeric receptors willhome to their target, will be stimulated by it to attract other effectorcells, or, by itself, will mediate specific destruction of the targetcells. In a preferred embodiment, the target cells are tumor cells andthe scFv domain is derived from an antibody specific to an epitopeexpressed on the tumor cells. It is expected that such anti-tumorcytolysis can also be independent of exogenous supply of IL-2, thusproviding a specific and safer means for adoptive immunotherapy.

[0064] In preferred embodiments, the immune cells are T-cells orNK-cells. The antibody scFvR design of the present invention will thusinvolve retargeting lymphocytes in vivo in an MHC-non-restricted manner.Thus, the T-cells can be re-targeted in vivo to tumor cells or any othertarget of choice toward which antibodies can be raised.

[0065] The term “single-chain Fv domain” is intended to include not onlythe conventional single-chain antibodies as described in references 16and 17, the entire contents of which are hereby incorporated herein byreference, but also any construct which provides the binding domain ofan antibody in single-chain form as, for example, which may include onlyone or more of the complementarity determining regions (CDRs), alsoknown as the hypervariable regions, of an antibody.

[0066] The gene encoding the transmembrane and cytoplasmic portions ofthe receptor molecule may correspond exactly to the natural gene or anygene which encodes the protein in its natural amino acid sequence.Furthermore, the present invention comprehends muteins characterized bycertain minor modifications to the amino acid structure of the molecule,such that the mutant protein molecules are substantially similar inamino acid sequence and/or 3D structure, and possess a similarbiological activity, relative to the native protein.

[0067] The transformed cells of the present invention may be used forthe therapy of a number of diseases. Current methods of administeringsuch transformed cells involve adoptive immunotherapy or cell-transfertherapy. These methods allow the return of the transformed immune systemcells to the blood stream. Rosenberg, S. A., Scientific American 62 (May1990); Rosenberg et al., The New England Journal of Medicine 323(9):570(1990).

[0068] The transformed cells of the present invention may beadministered in the form of a pharmaceutical composition with suitablepharmaceutically acceptable excipients. Such compositions may beadministered to any animal which may experience the beneficial effectsof the transformed cell of the present invention, including humans.

[0069] Those of ordinary skill in the art will further understand thatthe antibodies which are used to make the scFv portion of the presentinvention may be any antibody, the specificity of which is desired to betransferred to the immune cell. Such antibody may be against tumorcells, cells expressing viral antigens, anti-idiotypic oranti-clonotypic antibodies in order to specifically eliminate certainB-cells and T-cells, or antibodies against the constant region ofimmunoglobulin determinants. Thus, for example, if the antibody isspecific to the constant portion of IgE, it can serve to eliminateIgE-producing B-cells in order to alleviate allergy, etc. This list ofpossible antibodies is not intended to be exclusive and those ofordinary skill in the art will be aware of many additional antibodiesfor which important utilities exist upon combination with the receptorin accordance with the present invention.

[0070] The genes of the present invention can be introduced into theimmune cells by any manner known in the art, such as, for example,calcium phosphate transfection, electroporation, lipofection,transduction by retrovirus vector, use of a retroviral vector or a viralvector, etc.

[0071] The scFvR design is advantageous over the cTCR one. It requiresthe expression of only one gene instead of the gene pair required forthe cTCR, thereby providing simpler construction and transfection.

[0072] Furthermore, the scFvR design can be employed to confer antibodyspecificity on a larger spectrum of signaling molecules composed of onlyone chain. Additionally, the scFv maintains both V_(H) and V_(L)together in one chain; thus, even upon mixed pairing of chimeric withendogenous chains, the antigen-binding properties of the molecule areconserved. Finally, the fact that gamma and zeta constitute thesignaling chains of the TCR/CD3, the FcγRIII and the FcεRI expands thefeasibility of exploiting the chimeric receptor for retargeting otherhematopoietic cells, such as NK cells, basophils, or mast cells inaddition to T cells.

[0073] The chimeric scFvRγ of the invention or any of the simplemodifications thereof described below, that combine the specificity ofan antibody as a continuous single-chain and the effector function ofcytotoxic T cells and NK cells or regulatory function of helper T cells,constitute an important consequential development for targetedimmunotherapy. This approach exploits the scFv as theantigen-recognition unit and the potent cytotoxic responses of NK cellsand T cells and/or the ability of T cells to secrete lymphokines andcytokines upon activation at the target site, thus recruiting,regulating and amplifying other arms of the immune system.

[0074] The chimeric scFv receptors can confer on the lymphocytes thefollowing functions: antibody-type specificity toward any predefinedantigen; specific “homing” to their targets; specific recognition,activation, and execution of effector function as a result ofencountering the target; and specific and controlled proliferation atthe target site. Endowing the lymphocytes with an Fv from an antibodymay also serve for controlled and selective blocking of theaforementioned functions using soluble haptens or Fab′ of anti-idiotypicantibodies.

[0075] Candidate immune cells to be endowed with antibody specificityusing this approach are: NK cells, lymphokine-activated killer cells(LAK), cytotoxic T cells, helper T cells, and the various subtypes ofthe above. These cells can execute their authentic natural function andcan serve, in addition, as carriers of foreign genes designated for genetherapy, and the chimeric receptor shall serve in this case to directthe cells to their target. This approach can be applied also toanti-idiotypic vaccination by using helper T cells expressing chimericreceptors made of Fv of antiidiotypic antibodies. Such “designerlymphocytes” will interact and stimulate idiotype-bearing B cells toproduce antigen-specific antibodies, thus bypassing the need for activeimmunization with toxic antigens.

[0076] The invention will now be illustrated by the followingnon-limiting examples.

EXAMPLES Example 1 Constructions and Expression of the Chimeric ScFvRγ/ζChain Genes

[0077] In this example, the following materials and methods were used.

[0078] A. Cell lines and antibodies. MD.45 is a cytolytic T-lymphocyte(CTL) hybridoma of BALB/c mice allospecific to H-2^(b) (29). MD45.27J isa TCR α- mutant of MD.45. A.20 is a B lymphoma of BALB/c origin(ATCC#T1B 208). Cells were cultured in Dulbecco's modified Eagle'smedium (DMEM) supplemented with 10% fetal calf serum (FCS). Sp6, ananti-TNP mAb, and 20.5, an anti-Sp6 idiotype mAb, were provided by G.Kohler (30). Anti-human FcεRIγ chain polyclonal and monoclonal (4D8)(31) antibodies were provided by J. -P. Kinet and J. Kochan,respectively, and rabbit antibodies to murine zeta chain by M. Baniyash.

[0079] B. Constructions of chimeric genes. All the recombinant DNAmanipulations were carried out as described in updated editions ofSambrook et al. (1989) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor, N.Y., and Ausubel et al. (1987) Current Protocols inMolecular Biology, John Wiley & Sons. The specific genes encoding theV_(H) and V_(L) Of the Sp6 anti-TNP antibody were derived from thegenomic constructs described for the preparation of the cTCR (12, 32) byPCR amplifications using oligodeoxynucleotide primers designed accordingto the 5′ and 3′ consensus amino acid sequences of immunoglobulin Vregions (33) introducing the Xba I and BstEII restriction sites at theends of the scFv. In constructing the scFv we used theV_(L)-linker-V_(H) design containing a linker sequence similar to linker212 described by Colcher et al. (34). Accordingly, the V_(L)-3′ and theV_(H)-5′ primers include sequences comprising the 5′ and 3′ parts of thelinker, introducing Sal I in their 3′ and 5′ ends, respectively. Table Ilists the oligonucleotide primers used in the different constructions.In the examples, reference is made to the number of the specific primerused. Following digestion of the purified PCR products with Xba I andSal I (V_(L)) and Sal I and BstEII (V_(H)), the fragments were ligatedinto the Xba I and BstEII sites of a pRSV2neo-based expression vectorcontaining the leader of S1C5 kappa light chain (provided by S. Levy)and TCR constant region β chain (Cβ), prepared for the expression ofanti-38C.13 cDNA cTCR genes (12). The Cβ of this plasmid was thenreplaced with either the gamma chain amplified from a human cDNA clone(35) or the zeta chain amplified from Jurkat cDNA by using primersintroducing BstEII and Xho I at the 5′ and 3′ ends. A schematic diagramof the final scFvRγ expression vector is depicted in FIG. 2. Thesequences of the oligodeoxynucleotide primers used for the constructionof the chimeric scFvRγ and scFvRζ are delineated Table 1. TABLE IPrimers used for construction of the various scFvR Base code R = A or GS = C or G TACGTA SnaBI GTCGAC SaII GGATCC BamHI Y = C or T K = G or TCTCGAG XhoI GGTGACC BstEII AAGCTT HindIII W = A or T M = A or C TCTAGAXbaI GAATTC EcoRI GGTACC KpnI Primers for cDNA Reverse Transcription SEQID NO 1 mouse C_(γ)1 5′ GGCCAGTGGATAGAC 3′ SEQ ID NO 2 mouse kappa 5′GATGGTGGGAAGATG 3′ SEQ ID NO. 3 rat C_(γ)3 5′ CCATGRYGTATACCTGTGG 3′Primers for Single chain Fv SEQ ID NO 4 V_(κ)-5′ 5′CCCGTCTAGAGGAGAYATYGTWATGACCCAGTCTCCA 3′ SEQ.ID NO 5 V_(κ)-3′ 5′CCCGTCGACCCTTTWATTTCCAGCTTWGTSCC 3′ SEQ ID NO 6 V_(H)-5′ 5′CGGGTCGACTTCCGGTAGCGGCAAATCCTCTGAAGGCAAAGGTSAGG SEQ ID No 7 V_(H)-3′ 5′TGMRGAGACGGTGACCGTRGTYCCTTGGCCCCAG 3′ Receptor primers SEQ ID NO 8 5′ Cα(XhoI) 5′ CCTCGAGATAAAAAATATCCAGAACCCTGACCCTGCC 3′ SEQ ID NO 9 5′ Cα(BstEII) 5′ CGGTCACCGTCTCCTCAAATATCCAGAACCCTGACCCTGCC 3′ SEQ ID NO 10 5′Cβ (XhoI) 5′ CCTCGAGATAAAAGAGGACCTGAAAAACGTGTTCCCA 3′ SEQ ID NO 11 5′ Cβ(BstEII) 5′ CGGTCACCGTCTCCTCAGAGGACCTGAAAAACGTGTTCCCA 3′ SEQ ID NO 12 3′TCRα 5′ TACGTATCAGCTGGACCACAGCCGCAGCGTCAT 3′ SEQ ID.NO. 13 3′ TCRβ 5′TACGTATCAGCCTCTGGAATCCTTTCTCTTGAC 3′ SEQ ID NO 14 5′ FcRγ 5′CCGGTCACCGTCTCTTCAGCGGATCCTCAGCTCTGCTATATCCTGGA SEQ ID.NO. 15 3′ FcRγ 5′GGCAGCTGCTCGAGTCTAAAGCTACTGTGGTGG 3′ SEQ ID NO. 16 5′ TCRξ 5′GCTGGATCCCAAACTCTGCTACC 3′ SEQ ID.NO. 17 3′ TCRξ 5′CGCCTCGAGCTGTTAGCGAGGGGGC 3′ SEQ ID.NO. 18 5′ CD16 5′CCGGTCACCGTCTCCTCAGGGTACCAAGTCTCTTTCTGC 3′ SEQ.ID NO. 19 3′ CD16 5′CCCTCGAGTCATTTGTCTTGAGGGTC 3′ SEQ.ID.NO. 20 5′ IL-2Rβ 5′CCGGTCACCGTCTCTTCAGCGGATCCTACCATTCCGTGGCTCGGCCA SEQ.ID NO 21 5′ IL-2Rβ5′ GGAGATAGAAGCTTGCCAGG 3′ SEQ ID NO 22 3′ IL-2Rβ 5′CCTGGCAAGCTTCTATCTCC 3′ SEQ ID.NO 23 3′ IL-2Rβ 5′GGCTCGAGTCTACACCAAGTGAGTTGGG 3′

[0080] C. Expression of the Chimeric scFvRγ/ζ genes. Transfection of 20μg of pRSVscFvRγ/ζ DNA into 20×10⁶ MD.45 or MD.27J hybridoma cells wasperformed by electroporation using an ISCO power supply at 1.9 kV (32).Transfectants were selected in G418 at 2 mg/ml. Expression of scFvRγ/ζon the surface of transfected cells was evaluated by immunofluorescencestaining using the 20.5 anti-Sp6 idiotype and fluorescein-isothiocyanate(FITC)-labelled anti-mouse Fab′ antibody. Functional assays included anIL-2 production assay and a cytotoxicity assay in which the ability oftransfectants to respond specifically to TNP-modified A.20 target cellswas evaluated as detailed in Ref 9. The amount of IL-2 was determined byusing an IL-2-dependent CTL line and methyl tetrazolium acid (MTT)staining (36). Cytotoxicity assay was monitored by ⁵ ¹Cr release (29).All determinations were performed in triplicate.

[0081] D. Immunoprecipitation and Immunoblotting. Washed pelletscontaining 10⁸ cells were lysed in 1 ml of 1% digitonin, 0.12% TritonX-100 in 10 mM Tris.HCl—saline buffer pH 7.4 containing 10 mM EDTA, 1 mMphenylmethyl sulfonyl fluoride (Sigma), 10 μg/ml aprotinin and 10 μg/mlleupeptin (Boehringer Mannheim, GmbH). After 20 min at 0° C. andcentrifugation at 12000×g for 15 min, aliquots of the supernatants wereincubated with antibodies and then precipitated with second antibodiesand Protein G-Sepharose (Pharmacia) as described (32). Alternatively,cell lysates were mixed with sample buffer to a final concentration of1% NaDodSO₄ and either 10 mM iodoacetamide (for non-reducing gels) or 15mM dithiothreitol (for reducing gels). The washed immunoprecipitateswere dissociated in sample buffer under the same conditions. To avoiddestruction of the Sp6 idiotope, the samples were incubated at 20° C.for 30 min before NaDodSO₄/PAGE through 5-20% gel gradient. Separatedproteins were blotted onto nitrocellulose paper and allowed to reactwith anti-Sp6, anti-gamma or anti-zeta antibodies followed byperoxidase-labelled anti-immunoglobulin antibodies. Washed blots weredeveloped by using a chemiluminescence kit (ECL, Amsterdam) according tothe manufacturer's recommendations, and exposed to film (Kodak, X-OMATAR).

[0082] E. Results. To produce a chimeric receptor with an antigenbinding site of a given antibody and the signalling gamma or zetachains, we have adopted the scFv design (16, 17) which allows combiningboth entities into one continuous molecule. In engineering thepRSVscFvRγ/ζ expression vector (FIGS. 1,2), harboring the V_(L) andV_(H) of the Sp6 anti-TNP mAb (37), we introduced elements that enableits usage as a modular expression cassette to accommodate scFvs fromdifferent antibodies in combination with gamma, zeta or other chains.This was achieved by using oligonucleotide primers composed of sequencescommon to the majority of the 5′ and 3′ sequences of either V_(L) orV_(H) regions, flanked by relatively unique restriction sites, whichallow both in-frame ligation of the different units and its removal toother vectors (see Table I). We have chosen to use the5′-V_(L)-linker-V_(H)-3′ design, which was found suitable for theexpression of a variety of single-chain antibodies and their fragmentsin bacteria (17) , but the converse, 5′-V_(H)-linker-V_(L)-3′, alignment(16) can be used as well.

[0083] Introduction of the chimeric scFvRγ gene into the MD.45 murineCTL hybridoma (STA series of transfectants) or its MD45.27J TCRα-mutant, which does not express surface TCR/CD3 complex (STB series),resulted in the expression of the chimeric molecule on the cell surfaceof selected clones as revealed by staining with the anti-Sp6 idiotypicantibody (FIG. 3). Similar staining was observed for STZ, which wasderived by transfecting MD45.27J with the scFvRζ chimeric gene. Thesurface expression of the scFvRγ or scFvRζ molecule was independent ofthe TCR/CD3 complex; it did not restore surface expression of the CD3 inMD45.27J transfected STB or STZ cells, and some subclones of the STAthat initially expressed both scFvRγ and TCR/CD3 on their surface lost,upon a prolonged culture period, the TCR/CD3 expression without anyapparent effect on the scFvRγ expression and function (not shown).

[0084] Immunoblotting analysis of cell lysates prepared fromrepresentative STA and STB transfectants using either antiidiotypic mAb20.5 or polyclonal anti-human gamma antibodies, revealed 4 distinctbands of apparent molecular weight of 36, 54-62, 74-80 and 85-90 kDa,which did not appear in the parental cells (FIGS. 4A and 4B). Underreducing conditions one species, which corresponds to the predicted 36kDa monomeric form of the scFvRγ, was apparent, indicating themultimeric nature of the molecule. The band with apparent 75 kDamolecular weight corresponds to the homodimeric molecule, and the natureof the 90 kDa species is unknown. It might represent a novelgamma-associated polypeptide, analogous to the one recently reported(31). This species can be detected only in immunoblots of cell lysatesand is not apparent after surface iodination and immunoprecipitation(FIG. 5B), suggesting the intracellular origin of the molecule. Theappearance of bands in the range of 54-62 kDa was more pronounced in theSTB transfectant. It might represent heterodimers between the chimericscFvRγ chain and endogenous zeta and probably eta chains of the CD3complex. We therefore electrophoresed anti-Sp6 immunoprecipitates madefrom STB lysates, blotted the gels, and developed it with anti-Sp6,anti-gamma or anti-mouse zeta/eta antibodies (FIG. 5A). Both theanti-idiotypic and the anti-gamma antibodies revealed the four bandsfrom the transfected cells; however, the anti-zeta (which cross-reactswith the mouse eta chain) differentially developed only the 60 kDaspecies. Immunoprecipitation of surface-iodinated proteins with eitheranti-Sp6 or anti-gamma antibodies (FIG. 5B) demonstrates a main speciesof 75 kDa under non-reducing conditions. This is the homodimer of thechimeric chain.

Example 2 Expression of scFvRγ/ζ as Functional Receptors

[0085] To test whether the chimeric scFvRγ or scFvRζ can function as anactive receptor molecule, we studied the ability of the transfectedhybridomas to undergo antigen-specific stimulation. The MD.45 T cellhybridoma can be triggered through its TCR to produce IL-2, IL-3 orGM-CSF. It specifically recognizes and responds to H-2^(b) target cells(29), while its MD45.27J mutant cannot be stimulated through its TCR dueto the absence of an α chain. Upon introduction of the chimericSp6-scFvRγ, both of these cells could be specifically triggered toproduce IL-2 following incubation with TNP-modified stimulator cells(FIG. 6A) or plastic-immobilized TNP-fowl gamma globulin (TNP-FγG) (FIG.6B). Non-modified A.20 cells or FγG did not activate the transfectants,demonstrating the specificity of the response toward TNP. Stimulation ofthe various transfectants with immobilized antigen resulted in differentdegrees of reactivity. While STA responded to plastic-bound TNP-FγG inconsistent manner, STB and STZ (transfected with the scFvRγ and scFvRζ,respectively) lost their ability to undergo stimulation with immobilizedantigen but not with hapten-modified cells. Such behavior suggests thenecessity of additional synergistic signals for these cells. Indeed,costimulation with TNP-FγG plus either phorbol 12-myristate 13-acetate(PMA) or Ca⁺⁺ ionophore resulted in enhancement of IL-2 production (dataunshown). Incubation with soluble TNP-proteins even at highhapten/protein ratios did not result in activation but ratherspecifically inhibited triggering by immobilized antigen (FIG. 6B) orcell-bound hapten. The activation of GTAe.20, a transfectant expressinga two-chain chimeric TCR (9), was also inhibited by soluble TNF-FγG.Identical concentrations of antigen were needed to cause 50% inhibition(IC₅ ₀) of STA and GTAe.20 (FIG. 6B), indicating that the single-chainand the double-chain Fv display the same relative affinity to TNP.

[0086] Finally, we tested the ability of the chimeric receptors tomediate specific target cell lysis by incubating them with ⁵¹Cr labeledcells. As shown in FIGS. 7A and 7B, only the cells transfected with theSp6-scFvRγ or -scFvRζ could lyse TNP-modified target cells in adose-related fashion. This cytolytic activity was specific to TNP assoluble TNP-FγG blocked it (not shown) and unmodified A.20 cells werenot affected by the transfectants.

[0087] It is demonstrated here for the first time that a single-chain Fvof an antibody molecule fused to the gamma chain of the immunoglobulinFc receptor or to the zeta chain of the CD3 complex can be expressed inT cells as an antigen-specific receptor. The chimeric scFvRγ/ζ endowed Tcells with antibody-type specificity, transmitted a signal for IL-2production and mediated target cell lysis. The demonstration that thescFvRγ/ζ fusion protein could mediate antigen-specific stimulation of Tcells not expressing the TCR/CD3 receptor complex (as shown for the STBand STZ transfectants derived from the TCR-negative MD.27J mutant (FIGS.5A-5B and 6A-6B), strongly suggests that the gamma and zeta chains arecapable of autonomous activation of T cells. Yet, because of the lowlevel of heterodimers between the scFvRγ and the endogenous zeta and etachains (FIGS. 3 and 4A-4D), the possibility of some contribution by theresidual zeta (or eta) chain in the signalling process cannot beexcluded. Nonetheless, the present results clearly indicate that the TCRchains do not take part in this process, thus confirming andcomplementing recent observations in which antibody cross-linkingthrough the extracellular domains of CD4, CD8, IL-2 receptor, or CD16joined to the cytoplasmic tail of either one of the gamma/zeta familymembers resulted in T cell activation (26-28). Like scFvRγ/ζ chimericCD4 or CD16-gamma/zeta molecules expressed in cytotoxic lymphocytescould direct specific cytolysis against appropriate target cells (26,38). Analysis of mutations within the intracellular 18-residue motif,which has been recently assigned to account for the activity of thegamma/zeta chain, revealed that the ability to mediate calciumresponsiveness can be separated from the ability to support cytolysis(38). This opens new possibilities in which the chimeric chain, composedof scFv and genetically modified zeta or gamma chains can be used notonly to direct the specificity but also to dictate the selectedreactivity of lymphocytes.

[0088] The finding that immobilization of antigen is needed forefficient stimulation through scFvRγ/ζ and that soluble multimericligand (such as TNP-protein) did not trigger, but rather inhibited,reptor-mediated activation through cell- or plastic-bound TNP (FIG. 5B),indicates that mere engagement or even cross-linking of adjacent gammaor zeta chains does not result in T cell activation (as manifested byIL-2 release). The dependence on ligand immobilization for efficient Tcell triggering has been reported also for cTCR-mediated signalling (8,9), and the mechanisms underlying this are as yet unclear. Using thehybridoma transfected cells, different variants were obtained whichdiffer in their ability to respond to immobilized antigen or toTNP-modified stimulator cells of various origin. Because these variantsexpress surface receptors and respond to stimuli which bypass the TCR(such as with PMA+Ca++ ionophore), it was reasoned that they aredeficient in one of the components along the pathway leading to thecostimulatory signal, required for optimal cytokine release (39).Indeed, the fact that the addition of either PMA or ionomycin to theimmobilized antigen increased the response of most of these clones (notshown), strongly support this assumption.

Example 3 Targeting of Cytolytic Lymphocytes to Neu/HER2 ExpressingCells Using Chimeric Single-Chain Fv Receptors

[0089] Cell surface molecules essential for the transformed phenotype orgrowth of malignant cells are attractive targets for anti-cancerimmunotherapy. Antibodies specific to Neu/HER2, a humanadenocarcinoma-associated growth factor receptor, were demonstrated tohave tumor inhibitory capacity. Yet, the inefficient accessibility ofantibodies to solid tumor limits their clinical use. To redirecteffector lymphocytes to adenocarcinomas, we constructed and functionallyexpressed in T cells chimeric single-chain receptor genes incorporatingboth the antigen binding domain of anti-Neu/HER2 antibodies and the γ orζ signal transducing subunits of the T cell receptor/CD3 and theimmunoglobulin Fc receptor complexes. Surface expression of theanti-Neu/HER2 chimeric genes in cytotoxic T cell hybridomas endowed themwith specific Neu/HER2 recognition enabling their activation forinterleukin-2 production and lysis of cells overexpressing Neu/HER2.These chimeric genes can be used for the immunotherapy of cancer.

[0090] To establish the feasibility of the chimeric receptor approach toretarget cytolytic lymphocytes to tumor cells, we have usedanti-Neu/HER2 antibodies. The Neu/HER2 (also called c-erbB-2) is aprotooncogene product that encodes a growth factor receptor implicatedin the malignancy of several human adenocarcinomas that overexpress it.Out of a panel of monoclonal antibodies mAbs) specific to theextracellular portion of the Neu/HER2 protein (41), we selected mAb N29which significantly inhibited the tumorigenic growth of HER2/Neutransfected fibroblasts in nude mice, and induced phenotypicdifferentiation of various cultured breast cell lines (42). In thisexample, we show that T cells equipped with anti-Neu/HER2 specificity asthe ligand binding domain of the chimeric receptor, respond specificallyto Neu/HER2 bearing target cells.

[0091] In this example, the following materials and methods were used.

[0092] A. Cells and Antibodies. MD45 a murine allospecific CTL hybridoma(29) and MD45.27J, its mutant lacking the TCR a chain, served asrecipients for the chimeric genes. Stimulator and target cells used werehuman breast carcinoma cell lines SKBR3 and MDA 468, the human ovariancarcinoma cell line SKOV3, or HER2, a c-erbB-2 transfected 3T3-NIHfibroblasts (kindly provided by Dr. A. Ullrich). Cells were cultured inDMEM containing 10% FCS. N29 is a monoclonal anti-HER2 antibody (41),deposited with the Collection Nationale de Cultures de Microorganismes,Institut Pasteur, Paris France, on Aug. 19, 1992, under Registration No.CNCM I-1262. Anti-N29 idiotypic antiserum was prepared by immunizingrabbits with purified N29 protein and adsorption of the immune serum ona normal mouse Ig-agarose column. Rabbit anti-CD3ζ and anti-FcεRIγantibodies were kindly provided by Dr. J. -P. Kinet.

[0093] B. Construction and Transfection of Chimeric Genes. ChimericscFvN29Rγ or scFvN29Rζ genes were constructed from single chain Fv DNA(in the V_(L)-linker-V_(H) alignment), amplified by PCR from cDNAprepared of hybridoma producing the N29 anti-HER2 mAb, and either γ or ζgenes as described in Example 1 for the anti-TNP scFvR. The MD45 orMD45.27J hybridomas were transfected by electroporation with 20 μg ofDNA of pRSV2neo expression vectors harboring the chimeric genes and wereselected for growth in the presence of 2 mg/ml G-418 (GIBCO) for 2-3weeks as detailed in (9). Transfected cells were stained with eithercontrol serum or anti-N29 idiotypic antiserum (prepared by immunizingrabbits with purified N29 protein and adsorption of the immune serum ona normal mouse Ig-Agarose column. Following incubation at 4° C. with a1:200 dilution of sera, the cells were washed and treated withfluorescein isothiocyanate-labeled goat anti-rabbit antibody (JacksonLabs, West Grove, Pa., USA) for an additional hour at 4° C.Immunofluorescence of individual cells was determined with a FACSCAN(Becton Dickinson).

[0094] C. Detection of Soluble Receptor. Cell lysates were prepared fromthe transfectants by adding 100 μl of lysis buffer composed of 1% TritonX-100 in 0.15 M NaCl-10 mM Tris.HCl pH 7.4 buffer containing 10 mM EDTA,1 mM phenylmethyl sulfonyl fluoride (Sigma), 10 μg/ml aprotinin and 10μg/ml leupeptin (Boehringer Mannheim, GmbH) to a pellet of 5×10⁶ cells.After 30 min. at 0° C. and centrifugation, the nuclei-free supernatantwas added to wells of a microtitre plate precoated with 5 μg/well ofpurified HER2X protein. HER2X is a recombinant extracellular domain ofNeu/HER2 produced by CHO cells which were kindly provided by Dr. A.Ullrich. Following incubation for 2-4 hours at 4° C., plates were washedand incubated with 1 μg/ml of anti-human ζ or γ antibodies. Afterwashing and the addition of horseradish peroxidase labeled anti-Igantibodies (Jackson Labs), peroxidase substrate was added and the degreeof binding was determined by reading the OD₆ ₉ ₀.

[0095] D. IL-2 Production and Cytotoxic Assays. Stimulator cells(3×10⁴/well) were cultured in 96-well microculture plates for at least 6hours until adherent. For stimulation of transfectants with purifiedHER2X, wells of a microculture plate were coated with HER2X protein atthe indicated concentrations for at least 3 h at 22° C. and then washedtwice with medium. The transfected clones and their parental hybridomawere then added (10⁵/200 μl/well) in DMEM supplemented with 10% fetalcalf serum and 10⁻⁵ M of 2-β-mercaptoethanol. Following 20-24 hrs inculture, the amount of IL-2 produced was evaluated by the proliferationof the IL-2 dependent CTL-L cell line by the MTT calorimetric assay aspreviously described (9). To measure the cytotoxic activity, thetransfectants and their parental hybridomas were co-incubated with ⁵¹Crlabeled target cells at various effector to target ratios for 16 hrs.The ⁵¹Cr release assay was performed as described previously (16).

[0096] E. Results. Genes coding for single chain Fv of N29 fused toeither human γ or ζ chains were prepared in the pRSVscFvR vector, andused to transfect the murine MD45 allospecific CTL hybridoma or its TCRα-mutant MD45.27J which does not express the TCR/CD3 complex. Surfaceexpression of the chimeric chains on the hybridoma cells was detectedusing anti-idiotypic antibodies specific to the N29 anti-Neu/HER2 mAb(FIGS. 8A-8D). The integrity of the fusion protein comprising theantigen binding and signal transducing moieties was verified by areceptor-specific, enzyme-linked immunosorbent assay (ELISA), using arecombinant extracellular domain of Neu/HER2 (denoted HER2X) and anti-γand ζ antibodies. As shown in FIGS. 9A-9B, specific binding to HER2X wasobserved in whole cell lysates of the transfected but not of theuntransfected parental cells. Three transfectants, N29γ1 and N29γ15;both derived from MD45.27J cells transfected with the scFvRγ chimericgene, and N29ζM.1, a derivative of MD45 cells transfected with thescFvRζ chimeric gene, were selected for functional studies.

[0097] The single-chain chimeric receptor was found to transducespecific signals for T cell activation. Incubation of thescFvR-expressing cells together with human cancer cells, which expressNeu/HER2 on their surface, resulted in a marked activation as measuredby the production of IL-2 (FIG. 10A). This activation was mediated bythe scFvR and was Neu/HER2-specific, since cells which do notoverexpress Neu/HER2, like MDA-MB468 human breast carcinoma cells, didnot stimulate the production of high levels of IL-2, whereas cells thatdisplay large amounts of Neu/HER2, like the breast carcinoma SKBR-3cells, ovarian carcinoma SKOV-3 cells and an erbB-2 transfected murinefibroblast cell line, stimulated the hybridomas to produce high IL-2levels. Soluble, purified HER2X partially blocked the activation by thebreast carcinoma cells. However, upon immobilization, it served as apotent T cell activator, but only for the transfected cells (FIG. 10B).The T cell response to the immobilized antigen was in general weakerthan to the cellular targets. Possibly, co-stimulatory signals providedby accessory and adhesion molecules during T cell interactions mayamplify the intercellular interaction.

[0098] Finally, the ability of the transfected cells to mediate specifictarget cell killing was determined by the ⁵¹Cr release assay. When avariety of Neu/HER2 expressing cells were tested as targets (FIG. 11),we found that the HER2 cell line, an NIH-3T3 fibroblast overexpressingNeu/HER2, could serve as an adequate target. Following incubation withan scFvRγ-expressing T cell hybridoma (N29γ1) (FIG. 12), a substantiallevel of specific lysis was obtained. The scFvRζ expressing hybridoma(N29ζ18) gave only a marginal specific ⁵¹Cr release signal when comparedwith the untransfected hybridomas. The cytolytic effect wasNeu/HER2-specific, since untransfected NIH-3T3 fibroblasts did notundergo killing. Likewise, the parental MD45.27J cells did not cause anysignificant ⁵¹Cr release. The high levels of spontaneous ⁵¹Cr releasefrom several candidate human tumor lines that we tested, did not allowus to determine the killing potency in a reproducible manner.Nevertheless, in all experiments, the transfected cells induced asignificantly higher specific ⁵¹Cr release from human tumors (such asSKBR-3 breast and N87 gastric carcinoma cell lines, FIG. 11), than theparental hybridomas.

[0099] This study demonstrates that T cells expressing chimeric receptorgenes utilizing single-chain Fv of anti-tumor antibodies can beredirected to tumor cells. Binding of the scFvR to the tumor antigeneither in its isolated, immobilized form or in a cellular context wassufficient to trigger T cell activation and mediate target cell lysis.These results extend the previous examples using the anti-TNP scFvRγ/ζ.In all these instances, activated T cells or T cell lines have beenused.

Example 4 Functional Expression of scFvR With Anti-IgE Specificity

[0100] Allergic diseases are characterized by elevated synthesis of IgEupon stimulation by environmental allergens. The production of IgE isregulated by antigen specific helper and suppressor T cells. Tlymphocytes following activation, induce B cells to produce IgE. Thesecreted IgE binds preferentially to high affinity FCε receptors (FcεRI)on mast cells and basophils, thus sensitizing them. Followingencountering allergen the FcεRI-bound IgE is cross-linked and stimulatesexocytosis of granule-associated preformed pharmacologic mediators suchas histamine. Elimination of IgE producing cells can therefore terminateIgE production and thus prevent the onset of allergic responses. In thisexample, we take advantage of the fact that both IgE producing cells andtheir B-cell precursors express surface IgE and by employing the “Tbody” strategy using chimeric single-chain T cell receptor (scFvR)genes, made of an Fv of anti-IgE antibodies, we can specifically blockIgE production. The present example demonstrates the feasibility of thisapproach in an in vitro system, utilizing anti-mouse IgE antibodies.

[0101] In this example, the following materials and methods were used.

[0102] A. Generation and transfection of anti-IgE specific scFvR genes.The antibody which has been chosen is 84.1c, a rat mAb specific to anepitope on the murine Cε3 (43). The advantage of using 84.1c mAb is thatit reacts with free IgE and not with mast-cell bound IgE, thus it wasreasoned that it recognizes an epitope closely related to the FcεRIbinding site on IgE. The basic strategy for construction of the chimericgenes encoding the 84.1c mAb V_(L) and V_(H) in a continuous singlechain Fv linked to the constant region of the TCR α or β chains (Cα orCβ) is similar to the one described for the preparation of the anti-TNPscFvRγ/ζ chimeric genes and is schematically described in FIGS. 1 and 2.mRNA was selected on oligo (dT) cellulose from the 84.1c hybridoma.Single strand cDNA was synthesized using a 3′C_(κ) and 3′C_(H) heavyprimers employing M-MLV-reverse transcriptase (BRL). We amplified theV_(H) and V_(κ) by PCR using the mouse consensus oligonucleotide primerssimilar to the ones described above for the Sp6 anti-TNP scFv (44). TheV_(κ)-3′ primer and the V_(H) -5′ primer (5 and 6 of Table I) includedsequences comprising the 5′ and the 3′ parts of the linker, introducinga Sal I site in their 3′ and 5′ ends, respectively. Following digestionof the purified PCR product with Xba I and Sal I (V_(κ)) and Xba I andBstE II (V_(H)), the fragments were ligated and introduced into the XbaI and BstE II sites of pRSVL_(κ)Cα or Cβ expression cassettes. Theseexpression cassettes have been originally designed to express the doublechain chimeric TCR (cTCR) genes (45, 9, 32) and were constructed bycloning into the pRSV₂neo the leader of the 38c.13 κ-light chain 3′ tothe RSV LTR and downstream, either the Cα or Cβ of the human TCR. The Cαand Cβ were PCR-amplified from human TCR clones using primers 9 and 12from Table I for Cα and 11 and 13 for Cβ. Because we found previously(46) that the SRα promoter (47) drives transcription in T cells betterthan the RSV LTR, we adopted it here for the anti-IgE scFvR expression.For this purpose, we used the pBJ1neo plasmid (47). We cut out thecomplete scFv at the SnaBI sites from the pRSVscFvCβ/Cα vectors andintroduced it into the EcoRV of the pBJ1neo vector. FIG. 13 describesthe construction of the SRα based vector (pBJ-sc84.β).

[0103] The 84.1c based scFvCβ chimeric gene was introduced into eitherthe murine MD45 hybridoma (29) or the human Jurkat T cell leukemia β TCRnegative JRT3.5 mutant (48), respectively. Transfection was carried outby electrophoresis and transfectants were selected in the presence of 2mg/ml G418 as described in (9). JRT.T3.5 derived transfectants with thescFvR are denoted JSB and the MD.45 transfectants-JSMB.

[0104] B. Expression of the anti-IgE scFvR in T cells. To determine theintegrity of the chimeric genes, their ability to encode for surfacereceptor and to study the molecular nature of the receptor, we firsttransfected the Cβ based chimeric gene into the human leukemic Jurkatcell mutant JRT3.5, lacking the TCR β chain. In the absence ofanti-84.1c idiotype antibodies, we screened the transfectants obtainedfor the reappearance of surface CD3 by immunofluorescence, usinganti-CD3 antibodies, expecting that the chimeric scFvCβ chain willassociate with the endogenous TCR α chain and bring out the TCR/CD3complex. Although in parallel experiments we could bring out the CD3 tothe surface of JRT3.5 cells following transfection with V_(H)Cα orV_(L)Cα cTCR chains, we could not demonstrate any CD3-specific stainingof transfectants receiving the scFvCβ gene (data unshown). We thereforemonitored surface expression of the chimeric scFv genes in thetransfectants by rosette formation using trinitrophenylated sheep redblood cells (TNP-SRBC) coated with anti-TNP IgE SPE-7 (49). FIGS.14A-14E represents such experiments depicting the rosettes and showingthat they are specific to IgE and could be inhibited by adding IgE (andnot control IgG antibody, FIG. 14C). That the chimeric receptors containboth the antigen-binding moiety and TCR determinants in the samecomplex, was shown by analyzing lysates made of the transfectants.Incubation of such cell-free lysates on IgE-coated wells, followed bythe addition of anti-TCR-β specific mAbs and peroxidase labeledanti-mouse Ig antibodies, yielded specific binding (FIGS. 15A-15B).

[0105] C. Functional expression. Transfectants expressing chimericsurface receptors were tested for their ability to undergo specificactivation for IL-2 production following stimulation with IgE, eitherimmobilized by coating onto the plastic of the culture well or as asurface protein on IgE-producing hybridoma. FIG. 16 shows experiments inwhich the transfected cells were stimulated by plastic-bound IgE (oranti-CD3). It is clear that the Jurkat-derived transfectants, generatedby introduction of the scFvCβ, specifically produced IL-2. When we triedto stimulate the transfectants with the SPE-hybridoma cells, we foundthat soluble IgE secreted by these hybridomas blocked stimulation(exactly like in the 38C.13 system). We therefore fixed the IgEproducing hybridoma cells and indeed, as evident in FIG. 17, such cellsserved as potent stimulators.

[0106] Next we checked, using the cytotoxic MD45 T cell hybridomawhether the scFvCβ can arm and trigger cytotoxic cells to eliminate IgEproducing cells (known to express IgE on their surface). To mimic the invivo situation, as target cells we used murine splenic lymphocytes whichwere induced to produce IgE by culturing them in the presence oflipopolysaccharide (LPS) and IL-4. LPS+IL-4 are known to induce Ig classswitch in B cells and specifically trigger IgE and IgG₁ formation (50).In the experiments described in FIGS. 18A and 18B, we coincubated MD45transfectants expressing the anti-IgE scFvCβ with murine lymphocytes,added LPS+IL-4 and monitored both IgE and IgG accumulation into thesupernatants of these cultures. As shown in the figure, IgE secretionwas completely abrogated in cultures containing the scFvCβ T cells. Theeffect was very specific as no effect on IgG production could beobserved. The suppression of IgE production was most likely due toelimination of IgE producing cells by the redirected scFvCβ-bearing CTLhybridomas. The inability of control 84.1c B cell hybridoma to causesuch effect demonstrates that the lack of IgE accumulation in theculture medium is not because of passive absorption of IgE by the 84.1canti-IgE antibodies. This set of experiments clearly demonstrates thatcytotoxic T cells equipped with chimeric scFv-TCR can specificallyeliminate their target cells.

Example 5 Endowing Antibody Specificity to the Low Affinity FcγR(FcγRIII) Using Chimeric scFv Joined to the CD16α Chain

[0107] One of the most attractive candidates for the chimeric receptorapproach in natural killer (NK) cells is the low affinity receptor forIgG ((FcγRIIIA) which is composed of the ligand binding CD16αpolypeptide associated with the γ chain (51, 52). Triggering of NK cellsvia FcγRIII (either by anti-CD16 or through immune complexes) includescytokine production, expression of surface molecules and cytolyticactivity (53, 21). The CD16 polypeptide appears as membrane anchoredform in polymorphonuclear cells and as transmembrane form (CD16_(TM)) inNK (54). The FcγRIII-associated γ chain serves also as part of the FcεRIcomplex where it appears as homodimer, is very similar to the CD3 ζchain and can form heterodimers with it in some CTL and NK cells (52,21, 28, 23-25). Like ζ and η, chimeras between γ and CD4 directed CTL torecognize and kill cells expressing the HIV gp120 (26). Similar chimericreceptors between either the extracellular domain of CD8 (27) or Tac(28) in conjunction with γ, ζ or η have been recently reported instudies mapping the regions of these molecules which take part in thesignaling process.

[0108] It has been shown in previous examples that the binding domain ofa specific antibody in the form of an scFv can serve as the recognitionunit of the CD3 ζ (see also 44), TCR Cβ and FcεRI/FcγRIII γ (44). In thepresent example we report successful experiments in which we constructedand functionally expressed chimeric receptors composed of scFv andanti-TNP and the CD16α polypeptide of the FcγRIII.

[0109] In this example, the following materials and methods were used.

[0110] A. Design and construction of chimeric scFv-CD16α. For thescFv-CD16α design we have used the scFv of the Sp6 anti-TNP generatedbefore. The entire cytoplasmic and transmembrane and the immediateextracellular region (up to Gly206) of the CD16α (see FIG. 19) were PCRamplified from a human CD16α DNA clone (54), using the primers 18 and 19of Table I. The truncated CD16 DNA was inserted instead of the γ DNA inthe pRSVneoscFvRγ vector previously described.

[0111] B. Expression of the chimeric scFv-CD16α.

[0112] A. Expression in mast cells. Since the FcγRIII appears as aheterodimer complex consisted of CD16 and γ chains, to check theexpression of the chimeric scFvCD16 gene, we transfected it into the ratbasophilic leukemia (RBL) cell which is a mast cell expressingfunctional FcεRI (56). These cells produce excess of γ chain as part ofthe FcεRI and provide us with convenient function as thereceptor-triggered degranulation assay. Following electroporation of thechimeric scFv-CD16α as well as the scFvRγ and scFvRζ genes and selectionin G418, RBL clones were obtained which could be surface-stained by theanti-Sp6 idiotypic antibody. FIGS. 20A-20D shows the pattern of FACSanalysis of scFvCD16 transfected RBL and FIGS. 21A-21D shows thestaining of the scFvRγ and scFvRζ transfectants. Upon the addition ofTNP-protein conjugates to the scFvRγ and scFvRζ expressing RBLtransfectants, cross-linking of adjacent receptors by the multivalentantigen triggered degranulation as measured by specific release ofβ-hexoseaminidase to the supernatant (Table II). TABLE IIAntigen-Specific Degranulation of RBL Cells Transfected With ChimericscFvRγ And scFvRζ Genes Transfected Chain Stimulatory AntigenDegranulation % — IgE 3 — IgE + DNP − BSA 22 ScFvRγ IgE 3 scFvRγ IgE +DNP = BSA 31 scFvRγ TNP − BSA 50 scFvRζ IgE 3 scFvRζ IgE + DNP − BSA 38scFvRζ TNP − BSA 32

[0113] B. Expression in BW5147 cells. BW5147 is a murine thymoma whichdoes not express surface TCR/CD3 because it does not transcribe either γor ζ chains. As expected, transfection of the BW5147 cells with chimericscFvCD16 DNA did not yield any detectable surface receptor, yetintracellular receptor could be detected by immunoblotting of lysates(not shown). When the chimeric scFvCD16 and normal γ DNA wereco-electroporated into BW5147 cells, significantly high level of the Sp6idiotype could be detected on the surface of the transfectants asrevealed by immunofluorescence staining and FACS analysis (FIGS. 22A and22B). The transfectants responded to specific stimulus and produced IL-2following stimulation with TNP-modified A.20 cells or immobilizedTNP-fowl γ-globulin (TNP-FγG) (FIGS. 23 and 24A-24B).

[0114] Finally, we checked whether the chimeric scFvIL2R gene (made ofthe scFv of Sp6 and the β chain of the IL-2 receptor) can be expressedfollowing transfection on the surface of RBL cells. The Sp6-IL-2-Rchimeric gene was prepared by joining DNA containing the scFv of Sp6 toa 936 bp DNA segment cloned from PCR amplified DNA (using primers 20 and23 of Table I) containing the cytoplasmic and transmembrane regions(carboxy 312 amino acids) of the β-chains of the human IL-2 receptor.FIGS. 25A-25D shows the results of immunofluorescence staining of onesuch RBL transfectant with anti-Sp6 idiotypic antibodies. These resultsclearly demonstrate that the chimeric scFvIL2R can be expressed as asurface protein.

Example 6 Expression of Chimeric Single-Chain FV Receptors In BW5147Thymoma Functional Expression In BW5147 Thymoma Cells

[0115] BW5147 (BW) is a murine thymoma cell line which do not expressthe TCR or FcγR complexes (due to a defect in the ζ chain transcription(57)), and therefore served as a convenient cell-line to study theexpression of the different chimeric scFv receptors. Because BW cells donot produce endogenous ζ or γ chains, it is expected that followingtransfection, the chimeric receptors will be composed only of homodimersof the exogenous transgenes (in the case of scFvRγ or scFvRζ). Also, itprovides a system to study whether the chimeric scFvCD16 can beexpressed independently of γ or ζ chains.

[0116] The chimeric genes composed of an scFv of Sp6 anti-TNP mAb joinedto either one of the ζ, γ or CD16 chains were introduced byelectroporation into the BW cells and selected transfectants which grewin the presence of G-418 were analyzed for surface expression of the Sp6idiotope using the 20.5 anti-Sp idiotypic mAb. In parallel, a group ofBW cells was co-transfected with a mixture of scFvCD16+γ chain DNA. Theimmunofluorescence pattern of staining analysed by FACS is depicted inFIGS. 26A-26D. As can be seen, both BW.Sp6-γ and BW.Sp6-ζ transfectants(which received weither scFvRγ or scFvRζ DNA, respectively) could bespecifically stained with anti-Sp6 idiotypic antibody and thus express amoderate level of the chimeric receptor chains on their surface. Whenstudied for CD3 expression, using specific anti-CD3 mAb, we could notobserve any surface staining of the scFvRγ or scFvRζ transfectants (notshown), indicating that these chimeric genes are expressed on the cellsurface independently of the CD3 complex. None of the transfectantswhich was electroporated with scFvCD16 alone did express surfacereceptor (unshown). However, the co-transfection of scFvCD16 and the γchain DNA yielded transfectants, like the BW.Sp6-CD16 shown in FIGS.26A-26D, which express the chimeric receptors. These results clearlyprove that the CD16 chimeric chain was not sufficient for itself andneeded the γ chain for surface expression.

[0117] To study whether the chimeric receptors function in the BW cells,we tested the ability of transfectants to undergo stimulation for IL-2production following stimulation with TNP modified A.20 cells (FIG. 27)or immobilized TNP-FγG (FIG. 28). Although BW cells do not produce anyIL-2 following incubation with TNP-labeled antigen, the single-chainreceptor expressing transfectants produced IL-2 following stimulationwith either cellular or solid-phase antigen.

[0118] Taken together these studies demonstrate the appropriateexpression of the chimeric chains as functional receptors: they bindligand with antibody-type specificity on one end and signal for T cellstimulation on the other end. Although we have demonstrated hereexpression of the chimeric single chain receptors in non-TCR expressingT cells, it is reasonable to expect that natural killer cells, whichmake use of γ and CD16 in their signaling Fcγ receptor will behave in asimilar way.

Example 7 Transduction of Primary T-Cells with a Chimeric Single ChainFv Receptor Directed Against a Colon Cancer Antigen

[0119] The specificity of human tumor infiltrating lymphocytes (TIL) hasbeen redirected through modification of these cells with single chainantibody chimeric receptor genes linked to the γ subunit of the Fcreceptor (Hwu, P., et al. (1993) Journal of Experimental Medicine178:361-366, herein incorporated by reference). T-cells redirected withsingle chain antibody chimeric receptor genes (Hwu, P. et al. (1995)Cancer Research 55:3369 herein incorporated by reference) have beenfound to have in vivo antitumor activity. In this example, a chimericsingle chain T cell receptor made of an Fv of the GA733 antibody(Herlyn, D., et al. J. Immunol. Methods 73:157) and γ chain of the Fcreceptor is constructed and introduced into primary, anti-CD3 stimulatedperipheral blood lymphocytes (PBL) in order to redirect these T cellsagainst a colon cancer antigen.

Materials and Methods

[0120] Construction of GA733 chimeric receptor: Monoclonal antibody(MAb) GA733 (Herlyn, D., et al., (1984) J. Immunological Methods 73:157)binds to a 37 kD protein expressed on colorectal carcinoma cells. TotalRNA was prepared from a hybridoma cell line expressing MAb GA733(provided by Dorothee Herlyn, Wistar Institute), followed by reversetranscriptase polymerase chain reactin (RT-PCR) , using V_(H) and V_(L)primers corresponding to the 5′ and 3′ consensus amino acid sequences ofIg V regions. A scFv fragment was constructed as described (See FIG. 1),and was then joined to the γ chain of the Fc receptor to make chimericreceptor GA733-γ. The GA733-γ scFv receptor was then cloned into theSAM-EN retroviral vector, 5′ to the IRES (internal ribosomal entry site)to make SAM-G7γ-EN. The SAM-EN vector contains a splice acceptor (SA),which was PCR amplified from the 3′ terminus of the pol region fromwild-type moloney murine leukemia virus (MMLV; Treisman, J. et al.(1995), Blood 85:139). The SAM-EN vector contains an LTR promoter fromMMLV, as well as a neomycin phosphotransferase gene (Neo^(R)) 3′ to theIRES. The SAM-EN and SAM-G7γ-EN retroviral vectors were packaged intothe amphotropic packaging cell line PA317 (obtained from ATCC, CRL 9078)as previously described (Hwu, P. et al. (1993) Journal of Immunology150:4104, Hwu, P. et al. (1993) J. Exp. Med. 178:361, hereinincorporated by reference). Retroviral supernatant was harvested aspreviously described (Hwu, P. et al. (1993) J. Exp. Med. 178:361).

Transduction of PBL

[0121] Peripheral blood lymphocytes (PBL) were obtained from normaldonors. Mononuclear cells were isolated from buffy coats using aFicoll-Hypaque gradient, and were grown at 1.0×10⁶ cells per ml.Anti-CD3 (OKT3; Ortho Pharmaceuticals, Raritan, N.J.) was added to afinal concentration of 10 ng/ml on the initial day of culture only. PBLwere resuspended in retroviral supernatant supplemented with 8 μg/mlpolybrene and 600 International Units (IU) of interleukin-2 (IL-2) perml, and grown in 24-well plates containing 2 ml/well. Every 12 hours, 1ml of media was removed and replaced with 1 ml of freshsupernatant+polybrene/IL-2 for a total of 6 exposures to the retroviralsupernatant. 12 hours after the final exposure to supernatant, ⅔ of themedia was removed and replaced with RPMI containing 10% human AB serumand 600 IU IL-2/ml. 24 hours later (day 4 of culture), the PBL wereselected in the neomycin analog G418 (Geneticin; Life Technologies,Grand Island, N.Y.) for 5 days. 14 days after culture initiation, PBLwere tested for specific cytokine release and lysis.

Cytokine Assay

[0122] 2×10⁵ PBL were cocultured with 1.0×10⁵ tumor in 250 μl of medium(RPMI/10% human AB/600 IU/ml IL-2) for 24 hours at 37° C. Each samplewas performed in duplicate in flat-bottom 96-well plates. Supernatantswere then aspirated, centrifuged at 2,000 rpm to remove cells, decanted,and frozen at −70° C. Thawed samples were then tested in an ELISA forhuman GM-CSF, as previously described (Hwu, P. et al. (1993) J. Exp.Med. 178:361). As a positive control, PBL were incubated in wells coatedwith 2 μg/ml anti-CD3.

⁵¹Cr Release Cytotoxicity Assay

[0123] PBL were evaluated for their ability to lyse specific targetsusing a standard ⁵¹Cr release assay as described in Hwu, P. et al.(1993) J. of Immunology 150:4104, using ⁵¹-Cr-labeled targets.

Results

[0124] PBL transduced with the SAM-G7γ-EN receptor against colon cancerproduced large amounts of GM-CSF when co-cultured with colon cancer celllines CY13 and GREM, which express the GA733 antigen. In contrast, lessGM-CSF was produced by SAM-G7γ-EN upon coculture with the melanoma cellline 888 MEL or the erythroleukemia cell line K562. Nontransduced PBLand PBL transduced with the SAM-EN control retroviral vector did notproduce high amounts of GM-CSF in response to any of the targets. AllPBL groups produced high amounts of GM-CSF upon culture with the OKT3positive control (see Table III below). TABLE III GM-CSF Production byPBL PBL Responder [GM-CSF] pg/ml/5 × 10⁵ PBL/24 hours Stimulator NoneNontransduced Sam-EN Sam-G7γ-EN None 10 33 26 51 OKT3 0 7560 7990 8500K562 48 197 215 464 888 MEL 12 138 90 174 GREM 0 285 132 2976 CY13 197265 248 1282

[0125] TABLE IV Lysis of Colon Cancer Cell Line CY13 by PBL % SpecificLysis (Effector: Target) PBL 90:1 30:1 10:1 3.3:1 Nontransduced 14.3 8.4  4.8 1.3 SAM-EN 18.9 15.0 10.4 2.4 SAN-G7γ-EN 52.2 38.7 22.2 10.3

[0126] These results demonstrate that primary T-cells transduced with achimeric T-cell receptor against colon cancer can redirect those cellsto specifically lyse and secrete cytokine in response to colon cancercells.

[0127] All references cited herein, including journal articles orabstracts, published or corresponding U.S. or foreign patentapplications, issued U.S. or foreign patents, or any other references,are entirely incorporated by reference herein, including all data,tables, figures, and text presented in the cited references.Additionally, the entire contents of the references cited within thereferences cited herein are also entirely incorporated by reference.

[0128] Reference to known method steps, conventional methods steps,known methods or conventional methods is not in any way an admissionthat any aspect, description or embodiment of the present invention isdisclosed, taught or suggested in the relevant art.

[0129] The foregoing description of the specific embodiments will sofully reveal the general nature of the invention that others can, byapplying knowledge within the skill of the art (including the contentsof the references cited herein), readily modify and/or adapt for variousapplications such specific embodiments, without undue experimentation,without departing from the general concept of the present invention.Therefore, such adaptations and modifications are intended to be withinthe meaning and range of equivalents of the disclosed embodiments, basedon the teaching and guidance presented herein. It is to be understoodthat the phraseology or terminology herein is for the purpose ofdescription and not of limitation, such that the terminology orphraseology of the present specification is to be interpreted by theskilled artisan in light of the teachings and guidance presented herein,in combination with the knowledge of one of ordinary skill in the art.

REFERENCES

[0130] 1) Lowder, J. N. et al. Cancer Surv. 4:359-375 (1985).

[0131] 2) Waldmann, T. A. Science 252:1657-1662 (1991).

[0132] 3) Jain, R. K. J. Natl. Cancer Inst. 81:64-66 (1989).

[0133] 4) Mule, J. J. et al. Science 225:1487-1489 (1984).

[0134] 5) Rosenberg, S. A. et al. Science 233:1318-1321 (1986).

[0135] 6) Rosenberg, S. A. J. Clin. Oncol. 10:180-199 (1992).

[0136] 7) Kuwana, Y. et al. Biochem. Biophys. Res. Comm. 149:960-968(1987).

[0137] 8) Gross, G. et al. Transplant. Proc. 21:127-130 (1989).

[0138] 9) Gross, G. et al. Proc. Natl. Acad. Sci. USA 86:1002410028(1989).

[0139] 10) Becker, M. L. B. et al. Cell 58:911-921 (1989).

[0140] 11) Goverman, J. et al. Cell 60:929-939 (1990).

[0141] 12) Gross, G. Ph.D. thesis (Weizmann Institute of Science,Rehovot, Israel).

[0142] 13) Culver, K. et al. Proc. Natl. Acad. Sci. USA 88:31553159(1988).

[0143] 14) Kasid, A. et al. Proc. Natl. Acad. Sci. USA 87:473-477(1990).

[0144] 15) Adam, M. A. et al. J. Virol. 65:4985-4990 (1991).

[0145] 16) Huston, J. S. et al. Proc. Natl. Acad. Sci. USA 85:58795884(1988).

[0146] 17) Bird, R. E. et al. Science 242:423-426 (1988).

[0147] 18) Weissman, A. et al. EMBO J. 8:3651-3656 (1989).

[0148] 19) Bauer, A. et al. Proc. Natl. Acad. Sci. USA 88:3842-3846(1991).

[0149] 20) Unkeless, J. C. et al. Annu. Rev. Immunol. 6:251-281 (1988).

[0150] 21) Ravetch, J. V. and Kinet, J. -P. Annu. Rev. Immunol.9:457-492 (1991).

[0151] 22) Wirthmuller, U. et al. J. Exp. Med. 175:1381-1390 (1992).

[0152] 23) Orloff, D. G. et al. Nature (London) 347:189-191 (1990).

[0153] 24) Lanier, L. G. et al. J. Immunol. 146:1571-1576 (1991).

[0154] 25) Vivier, E. et al. J. Immunol. 147:4263-4270 (1991).

[0155] 26) Romeo, C. and Seed, B. Cell 64:1037-1046 (1991).

[0156] 27) Irving, B. A. and Weiss, A. Cell 64:891-901 (1991).

[0157] 28) Letourneur, F. and Klausner, R. D. Proc. Natl. Acad. Sci. USA88:8905-8909 (1991).

[0158] 29) Kaufmann, Y. et al. Proc. Natl. Acad. Sci. USA 78:2502-2506(1981).

[0159] 30) Rusconi, S. and Kohler, G. Nature (London) 314:330-334(1985).

[0160] 31) Schoneich, J. et al. J. Immunol. 148:2181-2185 (1992).

[0161] 32) Eshhar, Z. et al. in Practical Approach to Tumor Immunology,ed. G. Gallagher (IRL, Oxford) in press (1992).

[0162] 33) Kabat, E. A. et al. Sequences of Proteins of ImmunologicalImportance (Dept. of Health and Human Services, Washington, D.C.) 4thed. (1987).

[0163] 34) Colcher, D. et al. J. Nat. Cancer Inst. 82:1191-1197 (1990).

[0164] 35) Kuster, H. et al. J. Biol. Chem. 265:6448-6452 (1990).

[0165] 36) Mosmann, T. J. Immunol. Methods 65:55-63 (1983). 37) Kohler,G. and Milstein, C. Eur. J. Immunol. 6:511-519 (1976).

[0166] 38) Romeo, C. et al. Cell 68:889-897 (1992).

[0167] 39) Weaver, C. T. and Unanue, E. R. Immunol. Today 11:49 53(1990).

[0168] 40) Eshhar, Z. and Gross, G. Br. J. Cancer 62. (Suppl. X), 27-29(1990).

[0169] 41) Stancovski, I. et al. Proc. Natl. Acad. Sci. U.S.A.88:8691-8695 (1991).

[0170] 42) Bacus, S. et al. Cancer Res. 52:2580-2589 (1992).

[0171] 43) Schwarzbaum, S. et al. Eur. J. Immunol. 19:1915 (1989).

[0172] 44) Eshhar, Z et al. Proc. Natl. Acad. Sci. USA 90:720 (1993).

[0173] 45) Gross, G. and Eshhar, Z. FASEB 6:3370 (1992).

[0174] 46) Yablonski, D. “Transfection and functional expression of Tcell receptor genes”. M.Sc. Thesis presented to the Feinberg GraduateSchool, The Weizmann Institute of Science, Rehovot, Israel (1987).

[0175] 47) Lin, A. et al. Science 249:677 (1990)

[0176] 48) Weiss, A. and Stobo, J. J. Exp. Med. 160:1284 (1984).

[0177] 49) Eshhar, Z. et al. J. Immunol. 124:775 (1980).

[0178] 50) Snapper, C. et al. J. Immunol. 141:489 (1988).

[0179] 51) Lanier, L. et al. Nature 342:803 (1989).

[0180] 52) Anderson, P. M. et al. Proc. Natl. Acad. Sci. 87:2274 (1990).

[0181] 53) Cassatella, M. A. et al. J. Exp. Med. 169:548 (1989).

[0182] 54) Ravetch, J. V. and Ferussia, B. J. Exp. Med. 170:481 (1989).

[0183] 55) Lustgarten, J. and Eshhar, Z., in preparation.

[0184] 56) Barsumian, E. L. et al. Eur. J. Immunol. 11:317 (1981).

[0185] 57) Wegener, A. M. et al. Cell 68:83 (1992).

We claim:
 1. A chimeric gene comprising a first gene segment encoding asingle-chain Fv domain (scFv) of a specific antibody and a second genesegment encoding partially or entirely the transmembrane andcytoplasmic, and optionally the extracellular, domains of an immunecell-triggering molecule which, upon transfection to immune cells,expresses the antibody-recognition site and the immune cell-triggeringmoiety into one continuous chain.
 2. A chimeric gene according to claim1 wherein the second gene segment further comprises partially orentirely the extracellular domain of the immune cell-triggeringmolecule.
 3. A chimeric gene according to claim 1 wherein the first genesegment encodes the scFv domain of an antibody against tumor cells.
 4. Achimeric gene according to claim 1 wherein the first gene segmentencodes the scFv domain of an antibody against virus infected cells. 5.A chimeric gene according to claim 4 wherein the virus is HIV.
 6. Achimeric gene according to claim 1 wherein the second gene segmentencodes a lymphocyte receptor chain.
 7. A chimeric gene according toclaim 6 wherein the gene encodes a chain of the T cell receptor.
 8. Achimeric gene according to claim 7 encoding a subunit of the T cellreceptor.
 9. A chimeric gene according to claim 8 comprising a genesegment encoding the α, γ, γ or δ chain of the antigen-specific T cellreceptor.
 10. A chimeric gene according to claim 1 wherein the secondgene segment encodes a polypeptide of the TCR/CD3 complex.
 11. Achimeric gene according to claim 10 encoding the zeta or eta isoformchain.
 12. A chimeric gene according to claim 1 wherein the second genesegment encodes a subunit of the Fc receptor or IL-2 receptor.
 13. Achimeric gene according to claim 12 wherein the second gene segmentencodes a common subunit of IgE and IgG binding Fc receptors.
 14. Achimeric gene according to claim 13 wherein said subunit is the gammachain.
 15. A chimeric gene according to claim 14 comprising a genesegment coding for the CD16α chain of the FcγRIII or FcγRII.
 16. Achimeric gene according to claim 12 comprising a gene segment coding forthe α or β subunit of the IL-2 receptor.
 17. An expression vectorcomprising a chimeric gene according to claim
 1. 18. An immune cellendowed with antibody specificity transformed with an expression vectoraccording to claim
 17. 19. An immune cell endowed with antibodyspecificity comprising a chimeric gene according to claim
 1. 20. Animmune cell according to claim 19 selected from the group consisting ofa natural killer cell, a lymphokine activated cell, a cytotoxic T cell,a helper T cell and a subtype thereof.
 21. A primary T cell endowed withantibody specificity transformed with an expression vector according toclaim
 17. 22. A hematopoietic stem cell endowed with antibodyspecificity transformed with an expression vector according to claim 17.23. A tumor infiltrating lymphocyte cell endowed with antibodyspecificity transformed with an expression vector according to claim 17.24. A method of treatment of a tumor in a patient comprisingtransforming lymphocyte cells of the patient with an expression vectorcomprising a chimeric gene according to claim 1 in which the first genesegment encodes a scFv domain of an antibody directed against the tumorcells, and administering the transformed and thus activated cells to thepatient, said cells being targeted to the tumor cells thus causing tumorregression.
 25. A method according to claim 24 wherein peripheral bloodcells of the patient are transformed.
 26. A method according to claim 24wherein, hematopoietic stem cells of the patient are transformed.
 27. Amethod according to claim 24 wherein primary T cells of the patient aretransformed.
 28. Chimeric DNA sequence encoding a membrane-boundprotein, said chimeric DNA comprising in reading frame: a DNA sequenceencoding a signal sequence which directs the membrane bound protein tothe surface membrane; a DNA sequence encoding a non-MHC restrictedextracellular binding domain of a surface membrane protein which is asingle-chain antibody that binds specifically to at least one ligand,wherein said ligand is a protein on the surface of a cell or a viralprotein; a transmembrane domain from a protein selected from the groupconsisting of the CD3 zeta chain, the CD3 gamma chain, the CD3 deltachain and the CD3 epsilon chain; a cytoplasmic signal-transducing domainof a protein that activates an intracellular messenger system selectedfrom the group consisting of the CD3 zeta chain, the CD3 gamma chain,the CD3 delta chain, and the CD3 epsilon chain, wherein saidextracellular domain and cytoplasmic domain are not naturally joinedtogether and said cytoplasmic domain is not naturally joined to anextracellular ligand-binding domain, and when said chimeric DNA isexpressed as a membrane bound protein in a selected host cell underconditions suitable for expression, said membrane bound proteininitiates signalling in said host cell.
 29. A DNA according to claim 28wherein said extracellular domain is a single-chain antibody, or portionthereof containing ligand binding activity.
 30. A DNA sequence accordingto claim 28, wherein said single-chain antibody recognizes an antigenselected from the group consisting of viral antigens and tumor cellassociated antigens.
 31. A DNA sequence according to claim 28, whereinsaid single-chain antibody is specific for the HIV env glycoprotein. 32.A DNA sequence according to claim 28 where said cytoplasmic domain iszeta.
 33. A DNA sequence according to claim 28, wherein saidtransmembrane domain is naturally joined to said cytoplasmic domain. 34.An expression cassette comprising a transcriptional initiation region, aDNA sequence according to claim 28 under the transcriptional control ofsaid transcriptional initiation region, and a transcriptionaltermination region.
 35. An expression cassette according to claim 34,wherein said transcriptional initiation region is functional in amammalian host.
 36. A retroviral RNA or DNA construct comprising anexpression cassette according to claim
 35. 37. A cell comprising a DNAsequence according to claim
 28. 38. A cell according to claim 37,wherein said cytoplasmic domain is the CD3 zeta chain.
 39. A cellaccording to claim 37, wherein said cell is a mammalian cell.
 40. A cellaccording to claim 37, wherein said mammalian cell is a human cell. 41.A cell according to claim 37, wherein said cell is a hematopoietic stemcell.